Using Next-Generation Sequencing and Bioinformatic Methods to Predict New Genes That May Be Regulated by CD47 in Oral Squamous Cell Carcinoma

Round 1

Reviewer 1 Report

The authors have taken into account the previously raised issues and have modified accordingly this resubmitted version of their manuscript.

I think that the paper know is more clear and both study design and conclusions are more clear and sequentially reported. 

The only point is related to english language that in some points sounds incorrect.

Author Response

Reviewer #1: The authors have taken into account the previously raised issues and have modified accordingly this resubmitted version of their manuscript.

I think that the paper know is more clear and both study design and conclusions are more clear and sequentially reported.

The only point is related to english language that in some points sounds incorrect.

Response: Following the Reviewer’s comment, we have made the correction accordingly and manuscript has been edited by MDPI's English editing service(English-33274).

Reviewer 2 Report

The authors re-submited this manuscript to our journal without revision.

They don’t have been any positive-responses with our reviewers.

 

In my opinion, our journal should not deal this manuscript. Because, we decided this manuscript reject and our pure-review system have already done.

Author Response

Reviewer #2:

The authors re-submited this manuscript to our journal without revision.

They don’t have been any positive-responses with our reviewers.

In my opinion, our journal should not deal this manuscript. Because, we decided this manuscript reject and our pure-review system have already done.

Response: Following the Reviewer’s comment, we have incorporated all reviewers’ comments on the last revision. The following is a point-by-point response to the reviewers’ concerns.

Reviewer #1: The manuscript by Tseng and colleagues reports the use of RNA-Seq to identify genes and/or molecular pathways that may be regulated by CD47 in cell lines of oral squamous cell carcinoma.

Oral cell carcinoma is increasing both its incidence and mortality; moreover, it is often diagnosed in advanced stage, thus negatively impacting the overall patients' outcomes and survival. As a consequence, identify molecular biomarkers and pathways that may be used to improve the diagnostic strategies and/or to enhance the development of novel therapies, is an actual issue. In this context, investigate the effects of CD47 over-expression on mRNA profiling may be of interest. However, the study design need to be improved.

First, it is not clear why the 2 tested cell lines were chosen. Are these the only 2 models of OSCC? What are the difference between them? Are they currently used as OSCC in vitromodels?

Response: The Reviewer’s observation is correct, In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC).OECM-1, which are derived from surgical resection of a primary tumor from a Taiwanese patient with a history of betel-quid chewing. OC-2, which are derived from a primary tumor of the buccal mucosa of a Chinese patient with a habits of alcohol drinking, betel quid chewing and cigarette smoking[24, 25]. Therefore, that is the reason that we choose the 2 tested cell lines.

 

These aspects should be explained in the Introduction section. Moreover, the aims of the study, what is already known about CD47 expression in OSCCs and what issues authors plan to assess have to be clearly stated at the end of the Introduction.

Response: Following the Reviewer’s comment, we added the section in the introduction and end of the introduction.” In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23].” and “In the present study, we examine the effects of CD47 gene expression in  OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4) and lipid drop-associated proteins (HILPDA and PLIN2)were induced by CD47 and involved in the OSCC network.” 

Again, regarding the used cell lines and the methods used herein, what about their basal expression of CD47? The authors  transfected both of them in order to over-express CD47: not only they did not verified the efficiency of the performed transfection, but did not report CD47 basal level or the effect of endogenous CD47 silencing on gene expression. The methods used to carry out the RNA-Seq must be detailed better and also the bioinformatic tools used for data analyses and pathways prediction, and statistic tools need to be described under Methods.

Response: Following the Reviewer’s comment, we added the new results in the Fig. 1B. Overexpress of CD47 was performed by transfecting CD47 plasmid(1 ug) and CD47 expression was detected with qRT-PCR and compared to control. The results found that the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B). In addition, we know that silence CD47 affects downstream regulatory genes is very important, but the exploration of the mechanism is very important and complicated. Therefore, we will focus on the regulation of silence CD47 on downstream genes in more depth in the future. We also added the new bioinformatic tools in Methods. “2.5. Ingenuity® Pathway Analysis (IPA). IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses. Provide data classification and comprehensive explanation. In our study, IPA was used to conduct network analysis of CD47 downstream candidate genes and explore related signaling pathways information.” , “2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis. DAVID analysis is a powerful tool to the function of gene classification. Integrate data from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological processes. Calculate the similarity of the global annotation map through agglomeration algorithm and perform integrated classification according to biological functions, signal pathways and types of diseases.” and “ 2.7. Statistical analysis. The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells. All of the statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Software, CA).”

Section 3.1 has to be moved under Methods since it is just the description of the study design and not a result. The study design itself needs to be improved as specified above. Figure 1 may be useful for readers but its really too basic in its actual form.

Response: The Reviewer’s observation is correct, we modification the Fig 1A and added the new results in the Fig. 1B. Overexpress of CD47 was performed by transfecting CD47 plasmid(1 ug) and CD47 expression was detected with qRT-PCR and compared to control. The results found that the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B).

The other sections of Results have to be improved because are confused and difficult to follow.

Response: Following the Reviewer’s comment, we reversed Figure 4 and Figure 5 as well as adjust the order of Fig 4 A, B and C, and make the manuscript more easier to follow.

The discussion also should point on the potential translational values of the obtained results that does not clearly emerge.

Response: Following the Reviewer’s comment, the potential translational values of the obtained results were show in conclusions section. “These results provide important insights into possible new diagnostic genes for OSCC-targeting CD47. Although the mechanism by which CD47 regulates unfolded proteins and lipid drop-associated proteins is still unclear, the detailed signaling pathway will be discussed in more depth in the future.”

Minor Points:

Check the term "bioinformatics" throughout the text: if used as an adjective it must to be used in the singular (including the title).

After first mention within the text use just the abbreviation

Response: Following the Reviewer’s comment, we have made the correction accordingly.

 

Reviewer #2: This study provides important insights into possible new diagnostic strategies, including unfolded protein and lipid drop-associated protein for OSCC-targeting CD47.

Major comments:

1. The aims of this study was not mentioned in the end of the introduction. Please mention about it.

Response: Following the Reviewer’s comment, we added the section in the end of the introduction. “In the present study, we examine the effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4) and lipid drop-associated proteins (HILPDA and PLIN2)were induced by CD47 and involved in the OSCC network.” 

2. The introduction for CD47 was mentioned for a number of cancer but not for oral cancer. It should make a connection to oral cancer cells and becomes the aim of this study.

Response: Following the Reviewer’s comment, we added the new section and reference in the introduction. “ In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23].”

 

1. Moreover, some studies for investigating CD47 to oral cancer was not mentioned in oral cancer cells. For example,

(1) Oncol Lett 2018 Jun;15(6):9075-9080 (CD47 as a potential prognostic marker for oral leukoplakia and OSCC).

(2) Cells 2019, 8(12), 1658. (CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma)

Response: The Reviewer’s observation is correct, In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC).OECM-1, which are derived from surgical resection of a primary tumor from a Taiwanese patient with a history of betel-quid chewing. OC-2, which are derived from a primary tumor of the buccal mucosa of a Chinese patient with a habits of alcohol drinking, betel quid chewing and cigarette smoking[24, 25]. Therefore, that is the reason that we choose the 2 tested cell lines.

Reviewer #3: In this manuscript, the authors assay gene changes compared between oral squamous cell carcinoma (OSCC) cell and CD47 transfected OSCC cell.

I think the authors need to demonstrate in clinical data whether the genes they featured are associated with prognosis. Otherwise, most researcher will not be interested in the result.

Response: Following the Reviewer’s comment, we added the Fig 4C to verify the effect of four genes CD47, HSPA5, HYOU1, and PDIA4 in clinical data. “To further explore the role of CD47, HSPA5, HYOU1, and PDIA4 genes on cancer, we use human protein atlas (HPA) database[26] to analyze the expression of these target genes in cancer and their impact on survival ability. We know that OSCC occupies a high proportion of 90% of head and neck squamous cell carcinoma[27]. Therefore, in the HPA database, we select head and neck squamous cell carcinoma(499 patients sample) for gene expression and survival analysis. The results show that the fragments per kilobase per million(FPKM) of CD47, HSPA5, HYOU1, and PDIA4 was 22.19±10.51, 224.2±87.32, 25.35 ±13.45, 84.83±37.3, respectively. The increased expression of HSPA5 (cut off 25%), HYOU1(cut off 75%), and PDIA4(cut off 25%) was significantly associated with survival rate in 499 patient(Fig. 4C).

Reviewer 3 Report

The author have presented a paper about "Using next-generation sequencing and bioinformatic methods to predict new genes that may be regulated by CD47 in oral squamous cell carcinoma".

The topic is definitevely intersting however I have some major concerns as follows:

1) The authors used the OECM-1 and OC-2 cell lines: they should explain the reason for their choice

2) The authors were able to identify 14 genes involved in molecular functions, biological processes, and cellular components: in my view it could be an addition to spend a few words for each gene in the text to explain the role in signaling pathway (if known)

3) The discussion scetion is poor, please provide further details about the possible translational opportunities of your findings.

Author Response

Reviewer #3:

The author have presented a paper about "Using next-generation sequencing and bioinformatic methods to predict new genes that may be regulated by CD47 in oral squamous cell carcinoma".

The topic is definitevely intersting however I have some major concerns as follows:

1) The authors used the OECM-1 and OC-2 cell lines: they should explain the reason for their choice.

Response: The Reviewer’s observation is correct, In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC). OECM-1, which are derived from surgical resection of a primary tumor from a Taiwanese patient with a history of betel-quid chewing. OC-2, which are derived from a primary tumor of the buccal mucosa of a Chinese patient with a habits of alcohol drinking, betel quid chewing and cigarette smoking[24, 25]. Therefore, that is the reason that we choose the 2 tested cell lines.

2) The authors were able to identify 14 genes involved in molecular functions, biological processes, and cellular components: in my view it could be an addition to spend a few words for each gene in the text to explain the role in signaling pathway (if known)

Response: Following the Reviewer’s comment, we added the role of identify 14 genes in the Figure 3C.

3) The discussion scetion is poor, please provide further details about the possible translational opportunities of your findings.

Response: Following the Reviewer’s comment, we added the new discussion section in revision manuscript.

Reviewer 4 Report

Major point:

1) Does HSPA5 induce CD47 expression? Despite the authors have demonstrated that successful induction of HSPA5 expression (Figure 4G) and its effect on OECM-1 (Figure 4H) and OC-2 (Figure 4I) cell viability, it might be worth also quantifying CD47 expression in HSPA5-overexpressing OSCC cell lines.

2) The cell viability results (Figure 4H,I) are puzzling due to the two following reasons:

a) The y-axis in Figures 4H, I lacks a unit. For example, what does cell viability = 1.0 represents?

b) Given the observation that control cells display decreased viability in comparison to HSPA5-overexpressing cells, does HSPA5 simply promote cell proliferation or does it also inhibit cell death?

c) CCK-8 assay is not explained

Minor points:

1) "A total of 32 top-up and -down expression genes were listed, as well as 14 differentially expressed genes, which participated in DAVID biological process" (line 21) does not seem to make sense as it is not entirely clear how can a gene "participate in DAVID biological process"?

2) Please replace "IPA" with "Ingenuity Pathway Analysis (IPA)" (line 23).

3) "Human protein atlas (HPA) database to analyze gene expression and survival ability in human cancer" (line 24) lacks a verb. Please rephrase.

4) From "The results found that HSPA5, HYOU1 and PDIA4 were involved in the network and was highly expressed and mediated survival ability in cancer" (line 25) is not clear what network are the authors referring to?

5) It is not clear what the authors mean by "pa" in "Normally, oral cancer is a group of head and neck cancers, formed on the mucosal surface of the lips, hard pa, posterior molar triangle, and mandible" (line 37)?

6) "For example, CD47 is the ligand of SIRPα, which is a transmembrane receptor of the immunoglobulin (Ig) superfamily with extracellular immunoglobulin-like domains and antigen receptors of structural relationships; in addition, CD47 can also be used as a receptor of TSP-1, which is secreted by several non-hematopoietic cells such as platelets, monocytes, and macrophages" (line 53) is too long. Please split into two sentences.

7) "Related studies have shown that the expression of CD47 increases in migrating hematopoietic stem cells (HSCs), which manage to phagocytosis by phagocytes" (line 59) seems not to be grammatically correct with respect to "manage to phagocytosis by phagocytes" (line 59).

8) From "In ovarian cancer, an early study found that CD47 associates with CAR-T cells to inhibit tumor growth [20] and CD47 initiates an immune checkpoint through SIRPα signaling pathway in bladder cancer" (line 65) is not clear what the authors mean by "CAR-T cells" and where do these come from?

9) Please define abbreviation for "CAR-T" (line 66), "DGE" (line 109), "SE" (line 153), "RSEM" (line 183), "EBSeq" (line 183), "PPEE" (line 183), "CCK-8" (line 239).

10) It is not clear what "radioresistant" refers to in "Additionally, in radioresistant, we also found that the silencing of CD47 and HER2 can destroy breast cancer cells" (line 67)?

11) From "Additionally, in radioresistant, we also found that the silencing of CD47 and HER2 can destroy breast cancer cells" (line 67) is not clear how silencing of CD47 and HER2 destroys breast cancer cells?

12) Please change "destroy" to something like "eliminate" (line 68).

13) Please format "In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23]. In the present study, we examine the effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4))were induced by CD47 and involved in the OSCC network." (line 69) consistently with the rest of the text.

14) "In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues" (line 69) seems not to be grammatically correct with respect to "was detected in OSCC cell lines and significantly higher in OSCC than near tissues". Please fix.

15) Please change "expression" to "expression of" (line 69).

16) Please replace "pathway[23]" with "pathway [23]" (line 71).

17) Please change "examine" to "examined" (line 72).

18) Please change "OC-2 as well as" to "OC-2. We" (line 73).

19) Please change "(HSPA5, HYOU1, and PDIA4))were" to "(HSPA5, HYOU1, and PDIA4) are" (line 73).

20) Please replace "and involved in" with "within" (line 74).

21) Please describe the CCK-8 assay in the Materials and Methods section.

22) Please provide city name in France for the affiliation of Gibco in "The cancer cell lines were cultured in DMEM/F12 (Gibco, France) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin at 5% CO2 and in a 37 °C humidified incubator" (line 78).

23) Please replace "Transfection" with "transfection" (line 82).

24) Please change "pcDNA3.1-CD47(Addgene, #65473) and" to "pcDNA3.1-CD47 (Addgene, #65473) and" (line 84).

25) Please replace "(Addgene, #27165)were" with "(Addgene, #27165) were" (line 85).

26) Please replace "scientific, Waltham, MA USA" with "Fisher Scientific, Waltham, MA, USA" (line 85).

27) Please change "in vivo in a transfection" to "transfection" (line 86).

28) Please provide city and state affiliation for "Invitrogen" in "The total RNA was extracted from cell lines by the TRIZOL reagent (Invitrogen) and cDNA was synthesized using a cDNA Synthesis kit (Promega, Madison, WI, USA) according to manufacturer’s instructions" (line 90).

29) Please replace "of CD47, (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′)" with "for CD47 (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-93 TAA ACC AAG GCC AGT AG-3′)" (line 92).

30) From "The cDNA was PCR-amplified with the primers of CD47, (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′), HSPA5 (forward: 5’-CTG GCA AGA TGA AGC TCT CC-3’; reverse: 5’-AAA ACC CGA CAG AGG GAC AT-3’), HYOU1 (forward: 5’- GAC TTC GGC ATC TGA GTG GT-3’; reverse: 5’-GCT CCC AAG TCC ACC ATT AC-3’), PDIA4 (forward: 5’- GCT CAG CTC CAG GGA GAG -3’; reverse: 5’- GAT GAT CTC CAC CCA CCT GT-3’), B-actin (forward: 5’-ATG ATA TCG CCG CGC TCG TCG TC-3’; reverse: 5’-CGC TCG GCC GTG GTG GTG AA-3’) was used as the reference gene for normalization" (line 92) is not clear which genes (CD47, HSPA5, HYOU1, PDIA4, B-actin) were used for normalization?

31) "5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′" does not seem to be correct primer sequence notation with respect to "AT-TAA" (line 93).

32) Please change "B-actin" to "β-actin" (line 98).

33) "qPCR was analyzed by the ABI-7500 system (Applied Biosystems, Foster City, CA, USA) and the values were calculated using the 2−ΔΔCT method" is not semantically correct with respect to "qPCR was analyzed by" unless the authors are referring to analysis of the qPCR machine itself (line 100).

34) Please provide more specific and technically detailed information on how was RNA-seq quantification performed in the "2.4. RNA-seq quantification" Materials and Methods section.

35) Please provide city name in Taiwan for the affiliation of Illumina in "Differential gene expression was analyzed by RNA-seq quantification following the protocol of Illumina (Genomics, Taiwan), which obtained approximately 10 million reads for each sample" (line 104).

36) Please provide more specific and technically detailed information on how was IPA analysis performed in the "2.5. Ingenuity® Pathway Analysis (IPA)." Materials and Methods section. This needs to be relevant from the experimental point of view.

37) Please replace "Ingenuity® Pathway Analysis (IPA)." with "Ingenuity pathway analysis" (line 111).

38) Please format "Ingenuity® Pathway Analysis (IPA)." using italics (line 111).

39) "IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses" (line 112) sounds vague and rather irrelevant.

40) Please provide city name in USA for the affiliation of Ingenuity Systems in "IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses" (line 112).

41) Please change "CA" to "CA, USA" (lines 112, 128).

42) "Provide data classification and comprehensive explanation" (line 113) does not make sense. Please revise.

43) Please change "In our study, IPA" to "IPA" (line 114).

44) It is not exactly clear what the authors mean by "CD47 downstream candidate genes" in "In our study, IPA was used to conduct network analysis of CD47 downstream candidate genes and explore related signaling pathways information" (line 114)?

45) Please provide more specific and technically detailed information on how was DAVID analysis performed in the "2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis." Materials and Methods section. This needs to be relevant from the experimental point of view.

46) Please replace "(DAVID) analysis." with "analysis" (line 117).

47) Please format "2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis." using italics (line 117).

48) "DAVID analysis is a powerful tool to the function of gene classification" does not seem to make sense (line 119). What is "function of gene classification"?

49) In addition, "DAVID analysis is a powerful tool to the function of gene classification" is not grammatically correct with respect to "tool to the function of" (line 119).

50) "Integrate data from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological processes" (line  119) does not make sense.

51) Similarly, "Calculate the similarity of the global annotation map through agglomeration algorithm and perform integrated classification according to biological functions, signal pathways and types of diseases" (line 121) does not make sense.

52) Please change "analysis." to "analysis" (line 126).

53) Please format "2.7. Statistical analysis." using italics (line 126).

54) Please indent "The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells." on a new line (line 126).

55) "The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells" is not grammatically correct with respect to "expression level were" and "overexpression CD47 plasmid" (line 126). Please rephrase.

56) Please replace "plasmid(1ug, 24hr)" with "plasmid (1 μg, 24 hr)" (line 127).

57) Please change "All of the" to "All" (line 127).

58) Please provide city name in USA for the affiliation of GraphPad Software in "All of the statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Software, CA)" (line 127).

59) "Flowchart showing the gene expression profiling" does not seem to be a meaningful name for section 3.1. Please replace with a title that would reflect on the main finding in this subsection.

60) Please replace "overexpression in oral cancer" with "overexpression" (line 134).

61) From "In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC)" (line 135) is not clear what was the overall proportion of Asian patients enrolled in the study?

62) Please change "Asian who got the" to "Asian patients suffering from" (line 135).

63) Please replace "are" with "is" (lines 136, 138).

64) Please change "betel-quid" to "betel quid" (line 137).

65) Please change "habits" to "habit" (line 139).

66) Please replace "smoking[24,25].Therefore" with "smoking [24,25]. Therefore" (line 139).

67) Please remove "Therefore, that is the reason that we choose the 2 tested cell lines." (line 139).

68) The fact that Illumina NextSeq Sequencing was used is repeatedly stated in "Thus, this study used Illumina NextSeq Sequencing to analyze the genes of OECM-1 and OC-2 and whether they were 141 regulated by CD47" (line 140) and "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142). Please remove this redundancy.

69) Please change "Thus, this study" to "We" (line 140).

70) From "Thus, this study used Illumina NextSeq Sequencing to analyze the genes of OECM-1 and OC-2 and whether they were regulated by CD47" (line 140) is not evident the version of the Illumina NextSeq Sequencing used. Please fix.

71) Please replace "of OECM-1 and OC-2" with "in OECM-1 and OC-2 cells" or "in OECM-1 and OC-2 cell lines" (line 141).

72) "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142) is not a precise statement since Fig. 1A does not only show "that total RNA was extracted from the oral cancer cells". Please rephrase to summarize the overall meaning of Figure 1A.

73) "as per other bioinformatic studies" seems to be a redundant information in "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142). Please remove.

74) Please change "cells," to "cells" (line 142).

75) "In addition, the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B)" (line 143) does not make sense with respect to "the level of CD47 was induced by CD47 plasmid". Do the authors actually mean to say that CD47 was overexpressed?

76) Please replace "OC-2(Fig. 1B)" with "OC-2 cells (Fig. 1B)" or "OC-2 cell lines (Fig. 1B)" (line 145).

77) Please change "OECM-1(CD47/C)" to "OECM-1 (CD47/C)", "OC-2(CD47/C)" to "OC-2 (CD47/C)", "Gene Ontology (GO)" to "Gene ontology (GO)", and "DGE Report" to "DGE report" in Figure 1A.

78) The caption "Derived from Taiwanese patient" seems to clash with the black horizontal arrow in Figure 1A. Please fix.

79) Please indicate statistical significance in Figures 1B, 4E–I.

80) From the legend to Figure 1B is not clear what control was used for qRT-PCR?

81) From the legend to Figure 1B is not clear what is "ClusterProfiler"?

82) Please define abbreviation for "DGE" in the legend to Figure 1A.

83) Please replace "CD47-related" with "CD47" (line 148).

84) Please change "approaches.(B) Overexpress" to "approaches. (B) Overexpression" (line 151).

85) Please replace "transfecting CD47 plasmid(1 ug)" with "transfecting cells with CD47 plasmid (1 μg)" (line 151).

86) Please change "The potential gene expression was identified for CD47-mediated oral cancer" to something like "Gene expression profile in CD47-overexpressing oral cancer" (line 155).

87) Please replace "CD47 overexpression with" with "CD47-overexpressing" (line 158).

88) Please change "(Fig. 2A) in a volcano plot are shown in Fig. 2" to "(Fig. 2A)" (line 158).

89) Please replace "RNA-sequencing heatmap analysis showing genes" with "Genes" (line 159).

90) Please replace "-log10 (p-value)" with "-log10(P value)" (lines 160, 170).

91) Please remove italics formatting from "P" in "p-value" (lines 160, 170).

92) Please change "(Fig. 2B) were chosen for analyses" to "were chosen for further analyses (Fig. 2B)" (line 160).

93) From "Our results identified that the expression of 16 upregulated and 16 downregulated genes was found by CD47 overexpression in OECM-1/OC-2 compared to the control" (line 161) is not clear what control was used to compare gene expression following CD47 overexpression?

94) Please replace "found by" with "found to be associated with", "found to be modulated by" or "found to be regulated by" (line 162).

95) Please change "OECM-1/OC-2" to "OECM-1 and OC-2 cells" or "OECM-1 and OC-2 cell lines" (lines 162, 195). 

96) Please replace "(Fig. 2C). Table 1 lists the 32 association genes." with "(Fig. 2C) (Table 1)" (line 162).

97) Please change "CD47 overexpression on" to "CD47-overexpressing" (lines 166, 168).

98) Please replace "genes, showing that CD47 mediated 617 genes that were upregulated and 653 genes that were downregulated" with "genes show that 617 genes were upregulated and 653 genes were downregulated" (line 171).

99) From Table 1 is not clear what does "M1-C" refer to? Please explain in a legend.

100) From Table 1 is also not clear why is luteolin compared to control?  Please explain the experimental design for luteolin use in a legend. Also, please specify the control.

101) Lastly, from Table 1 is not evident what does "head to head" (SMC2-AS1) and "readthrough" (TRIM6-TRIM34) indicate? Please explain in a legend.

102) Please define abbreviation for "NA" in the legend to Table 1.

103) Please change "changes by the overexpression oof" to "changes induced by the overexpression of" (line 175).

104) Please replace "(DEGs) in CD47-mediated OSCC" with "in CD47-overexpressing oral squamous cell carcinoma" (line 179).

105) Please change "analysis, with a Venn diagram showing the number" to "analysis" (line 181).

106) Please replace "oral cancer (OECM-1/OC-2)" with "OECM-1 and OC-2 oral cancer" (line 184).

107) Please change "(Fig. 3A), Furthermore" to "(Fig. 3A). Furthermore" (line 184).

108) Please replace "demonstrate" with something like "display" or "plot" (line 185).

109) Please change "(Fig. 3B), with Fig. 3C presenting the 14 association genes" to "(Fig. 3B,C)" (line 185).

110) It is not clear what do the numbers in brackets indicate in Figure 3A? Please explain in the respective figure legend.

111) Please flip horizontally Figure 1A so that OECM-1 circle is on the left and OC-2 on the right.

112) Analogously, flip horizontally Figure 1B so that OC-2 control panel is on the left and OC-2-CD47 on the right.

113) The meaning of the heatmap is not clearly defined in Figure 1B. What is the parameter displayed and what does yellow and dark blue color indicate? Please explain to the readers.

114) It is not clear what does the horizontal and vertical cladogram indicate in Figure 3B? Please explain in the respective figure legend.

115) From Figure 3C is not clear what does "NMD candidate" (NPHP3-ACAD11, RTEL1-TNFRSF6B) and "readthrough" (PHOSPHO2-KLHL23, TRIM6-TRIM34) refer to? Also, please define abbreviation for "NMD" in the respective figure legend.

116) Please replace "Gene Symbol" with "Gene symbol", "Official Gene Symbol" with "Official gene symbol", "Gene Name" with "Gene name", "cell lines" with "Cell line", and "OECM-1/ OC-2" with "OECM-1/ OC-2" 7x in Figure 3C.

117) Please replace "function of DEGs on CD47 overexpression in" with "function of differentially expressed genes in CD47-overexpressing" or "predicted function of differentially expressed genes in CD47-overexpressing" (line 190).

118) Please change "comparison" to "comparison was" (line 191).

119) Please replace "upregulated/downregulated genes" with "DEGs" (line 194).

120) Please change "We also" to "In addition, we" (line 197).

121) Please replace "by CD47 overexpression in the" with "CD47-overexpressing" (line 200).

122) Please change "appeared in the MFs, BPs, and CCs (Fig. 4B)" to "appear in all three categories" (line 206).

123) Please replace "on" with "in" (line 207).

124) Please change "use" to "used" (line 208).

125) Please replace "survival ability" with "on the ability of cells to survive" (line 209).

126) Please change "carcinoma[27]" to "carcinoma [27]" (line 210).

127) Please replace "select" with "selected" (line 211).

128) Please change "carcinoma(499 patients sample)" to "carcinoma (499 patients sample)" (line 211).

129) Please format "pression and survival analysis. The results show that the fragments per kilobase per mil-" (line 212) consistently with the rest of the text.

130) Please replace "million(FPKM)" with "million (FPKM)" (line 212).

131) Please change "22.19±10.51, 224.2±87.32, 25.35±13.45, 84.83±37.3" to "22.19 ± 10.51, 224.2 ± 87.32, 25.35 ± 13.45, 84.83 ± 37.3" (line 213).

132) From "The increased expression of HSPA5 (cut off 25%), HYOU1(cut off 75%), and PDIA4(cut off 25%) was significantly associated with survival rate in 499 patient(Fig. 4C)" (line 214) is not clear what the authors mean by "cut off"?

133) Please replace "HYOU1(cut off 75%), and PDIA4(cut off 25%)" with "HYOU1 (cut off 75%), and PDIA4 (cut off 25%)" (line 215).

134) Please change "patient(Fig. 4C)" to "patients (Fig. 4C)" (line 216).

135) From "In addition, HSPA5 was highly expressed in cancer and positively and significantly correlated with CD47 (R=0.099, P = 0.027, Fig. 4D)" (line 216) is clear what does R refer to?

136) Although the authors claim that "In addition, HSPA5 was highly expressed in cancer and positively and significantly correlated with CD47 (R=0.099, P = 0.027, Fig. 4D)" (line 216), the correlation coefficient value is only 0.099 (very weak or no association). Please resolve this dichotomy by acknowledging either the low level of correlation or the absence of it in the text.

137) Please change "R=0.099" to "R = 0.099" (line 217).

138) Please replace "In addition to prove HSPA5 were" with "To prove that HSPA5 was" (line 218).

139) Please change "OECM-1/OC-2" to "OECM-1 and OC-2" (lines 219, 224, 245).

140) Please replace "OECM-1(Fig. 4E) and OC-2(Fig. 4F) OSCC cell lines" with "OECM-1 (Fig. 4E) and OC-2 (Fig. 4F) cell lines" (line 220).

141) Please change "verify" to "verified" (line 221).

142) Please replace "HSPA5" with "HSPA5 overexpression (line 221).

143) Please replace "lines," with "lines." (line 221).

144) Please change "transfer to cell lines and the gene overexpression" to "transfected into the cells and HSPA5 expression" (line 222).

145) Please replace "qRT-PCR(Fig. 4G)" with "qRT-PCR s(Fig. 4G)" (line 223).

146) Please change "The cell" to "Cell" (line 223).

147) Please replace "found" with "revealed" (line 223).

148) From "these data suggests that HSPA5, HYOU1 and PDIA4 was significantly associated with survival rate and HSPA5 was suitable prognostic markers of CD47 mediated in cancer" (line 224) is not clear what the authors mean by "CD47 mediated in cancer" (line 224)?

149) Please change "these data suggests" to "These data suggest" or to something like "Altogether, these data suggest" (line 224).

150) Please replace "PDIA4 was" with "PDIA4 were" (line 225).

151) Please change "HSPA5 was" to "HSPA5 is a" (line 225).

152) Please replace "markers" with "marker" (line 226).

153) Figure 4A is rather pixelated, please enlarge and/or increase its resolution.

154) Please replace "Focus molecular" to "Molecular focus", "SDF2L1/HSPA5/CALR/HYOU1/SESN2/PDIA4/" with "SDF2L1, HSPA5, CALR, HYOU1, SESN2, PDIA4", "CHAC1/CTH/ DDIT3/HERPUD1/HSPA5/SESN2/XBP1/SDF2L1/DERL3/ ASNS/HYOU1/CALR/PDIA4" with "CHAC1, CTH, DDIT3, HERPUD1, HSPA5, SESN2, XBP1, SDF2L1, DERL3, ASNS, HYOU1, CALR, PDIA4", and "HSPA5/SDF2L1/CALR/SLC3A2/SLC1A5/PDIA4/SLC1A4/HYOU1" with "HSPA5, SDF2L1, CALR, SLC3A2, SLC1A5, PDIA4, SLC1A4, HYOU1" in Figure 4A.

155) From Figure 4A is not clear what the authors mean by "Focus molecular"? Please explain in the respective figure legend.

156) Please remove the two small black lines present under the label "B" in Figure 4B.

157) Please remove "Number of values: Sample number" from Figure 4C.

158) Please change "Fragments Per Kilobase of transcript per Million mapped reads (FPKM)" to "Fragments per kilobase of transcript per million mapped reads (FPKM)" in Figure 4C.

159) Please define abbreviation for "CI" under the table in Figure 4C.

160) From the legend to Figure 4C is not clear what does "Number of values" exactly refer to as "Number of values: Sample number" does not provide much clue?

161) Please replace "(B)Three" with "(B) Three" (line 233).

162) Please change "OSCC.(C, D)" to "OSCC. (C, D)" (line 235).

163) "The gene expression and survival rate was analyzed by FPKM of CD47, HSPA5, HYOU1 and PDIA4 through HPA database" (line 235) does not seem to make sense with respect to "gene expression and survival rate was analyzed by FPKM". Please rephrase.

164) Please format "The gene expression and survival rate was analyzed by FPKM of CD47, HSPA5, HYOU1 and PDIA4 through HPA database. (E,F)" (line 235) consistently with the rest of the text.

165) Please replace "OECM-1 and OC-2 cells overexpressed with CD47" with "CD47-overexpressing OECM-1 and OC-2 cells" (line 337).

166) Please change "qRT-237 PCR.(G)HSPA5" to "qRT-237 PCR. (G) HSPA5" (line 237).

167) Please format "gene expression was analyzed by qRT-PCR and the (H, I)cell growth was analyzed by CCK-8 assay." (line 238) consistently with the rest of the text.

168) Please replace "and the (H, I)cell" with "and (H, I) cell" (line 238).

169) From "*P < 0.05 vs. untreated control; two-tailed Student’s t-test" (line 239) is not clear what the authors refer to by "untreated control" as there were not drugs indicated that the cells were treated with?

170) Please format "*P < 239 0.05 vs. untreated control; two-tailed Student’s t-test." (line 239) consistently with the rest of the text.

171) Please change "DEGs in CD47 overexpression" to "differentially expressed genes following CD47 overexpression" or "differentially expressed genes in CD47-overexpressing oral squamous cell carcinoma" (line 242).

172) "We also used DAVID biological process analysis to analyze the function of the DEGs with CD47 overexpression in the OECM-1 and OC-2 cell lines from RNA sequencing" does not seem to make sense with respect to "function of the DEGs with CD47 overexpression in the OECM-1 and OC-2 cell lines from RNA sequencing" (line 243). Please revise.

173) Please replace "with CD47 overexpression in the" with "CD47-overexpressing" (line 244).

174) Please change "separately concluded" to "concluded" (line 245).

175) Please replace "to endoplasmic" with "endoplasmic" (line 247).

176) Please change "to unfolded" to "unfolded" (line 248).

177) Please replace "OECM-1" with "OECM-1 cells" (line 250).

178) Please change "OC-2" with "OC-2 cells" (line 251).

179) Please replace "41genes" with "41 genes" (line 251).

180) Figure 5 is rather pixelated, please enlarge and/or increase its resolution.

181) From the legend to Figure 5 is not clear, what the numbers associated with different processes indicate?

182) Please change "on CD47 overexpression in the" to "in CD47-overexpressing" (line 258).

Author Response

Reviewer #4:

Major point:

1) Does HSPA5 induce CD47 expression? Despite the authors have demonstrated that successful induction of HSPA5 expression (Figure 4G) and its effect on OECM-1 (Figure 4H) and OC-2 (Figure 4I) cell viability, it might be worth also quantifying CD47 expression in HSPA5-overexpressing OSCC cell lines.

Response: The Reviewer’s comment is well taken. CD47 expression was induced by HSPA5-overexpressing in cell lines (data not show). Previous study also demonstrated that inhibition of glucose-regulated protein-78 (GRP78), a member of HSP70 family, downregulated CD47 expression in tumor cells (PMCID: PMC5557514). Therefore, we believed that CD47 and HSPA5 have a feedbackloop in OSCC cell lines.

2) The cell viability results (Figure 4H,I) are puzzling due to the two following reasons:

1. a) The y-axis in Figures 4H, I lacks a unit. For example, what does cell viability = 1.0 represents?

Response: Following the Reviewer’s comment, we added the unit in y-axis of Figures 4H, I.

1. b) Given the observation that control cells display decreased viability in comparison to HSPA5-overexpressing cells, does HSPA5 simply promote cell proliferation or does it also inhibit cell death?

Response: Following the Reviewer’s comment, cell viability was analyzed by CCk-8 assay in the Figures 4H and I. CCK-8 allows sensitive colorimetric assays for the determination of the number of viable cells in the proliferation and cytotoxicity assays. In discussion section” knockout mouse models have demonstrated that HSPA5 is required for cell proliferation and is critical for embryonic cell growth and survival [34]. In vitro and in vivo study found that silencing of HSPA5 expression in cancer cells inhibits tumor cell metastasis [35]. Therefore, we believe that HSPA5 plays an important role in cancer progression.” Based on the above conclusions, we suggested that HSPA5 can promote cell proliferation.

1. c) CCK-8 assay is not explained

Response: Following the Reviewer’s comment, CCK-8 assay was described in the Materials and Methods section.

Minor points:

1) "A total of 32 top-up and -down expression genes were listed, as well as 14 differentially expressed genes, which participated in DAVID biological process" (line 21) does not seem to make sense as it is not entirely clear how can a gene "participate in DAVID biological process"?

2) Please replace "IPA" with "Ingenuity Pathway Analysis (IPA)" (line 23).

3) "Human protein atlas (HPA) database to analyze gene expression and survival ability in human cancer" (line 24) lacks a verb. Please rephrase.

4) From "The results found that HSPA5, HYOU1 and PDIA4 were involved in the network and was highly expressed and mediated survival ability in cancer" (line 25) is not clear what network are the authors referring to?

5) It is not clear what the authors mean by "pa" in "Normally, oral cancer is a group of head and neck cancers, formed on the mucosal surface of the lips, hard pa, posterior molar triangle, and mandible" (line 37)?

6) "For example, CD47 is the ligand of SIRPα, which is a transmembrane receptor of the immunoglobulin (Ig) superfamily with extracellular immunoglobulin-like domains and antigen receptors of structural relationships; in addition, CD47 can also be used as a receptor of TSP-1, which is secreted by several non-hematopoietic cells such as platelets, monocytes, and macrophages" (line 53) is too long. Please split into two sentences.

7) "Related studies have shown that the expression of CD47 increases in migrating hematopoietic stem cells (HSCs), which manage to phagocytosis by phagocytes" (line 59) seems not to be grammatically correct with respect to "manage to phagocytosis by phagocytes" (line 59).

8) From "In ovarian cancer, an early study found that CD47 associates with CAR-T cells to inhibit tumor growth [20] and CD47 initiates an immune checkpoint through SIRPα signaling pathway in bladder cancer" (line 65) is not clear what the authors mean by "CAR-T cells" and where do these come from?

9) Please define abbreviation for "CAR-T" (line 66), "DGE" (line 109), "SE" (line 153), "RSEM" (line 183), "EBSeq" (line 183), "PPEE" (line 183), "CCK-8" (line 239).

10) It is not clear what "radioresistant" refers to in "Additionally, in radioresistant, we also found that the silencing of CD47 and HER2 can destroy breast cancer cells" (line 67)?

11) From "Additionally, in radioresistant, we also found that the silencing of CD47 and HER2 can destroy breast cancer cells" (line 67) is not clear how silencing of CD47 and HER2 destroys breast cancer cells?

12) Please change "destroy" to something like "eliminate" (line 68).

13) Please format "In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23]. In the present study, we examine the effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4))were induced by CD47 and involved in the OSCC network." (line 69) consistently with the rest of the text.

14) "In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues" (line 69) seems not to be grammatically correct with respect to "was detected in OSCC cell lines and significantly higher in OSCC than near tissues". Please fix.

15) Please change "expression" to "expression of" (line 69).

16) Please replace "pathway[23]" with "pathway [23]" (line 71).

17) Please change "examine" to "examined" (line 72).

18) Please change "OC-2 as well as" to "OC-2. We" (line 73).

19) Please change "(HSPA5, HYOU1, and PDIA4))were" to "(HSPA5, HYOU1, and PDIA4) are" (line 73).

20) Please replace "and involved in" with "within" (line 74).

21) Please describe the CCK-8 assay in the Materials and Methods section.

22) Please provide city name in France for the affiliation of Gibco in "The cancer cell lines were cultured in DMEM/F12 (Gibco, France) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin at 5% CO2 and in a 37 °C humidified incubator" (line 78).

23) Please replace "Transfection" with "transfection" (line 82).

24) Please change "pcDNA3.1-CD47(Addgene, #65473) and" to "pcDNA3.1-CD47 (Addgene, #65473) and" (line 84).

25) Please replace "(Addgene, #27165)were" with "(Addgene, #27165) were" (line 85).

26) Please replace "scientific, Waltham, MA USA" with "Fisher Scientific, Waltham, MA, USA" (line 85).

27) Please change "in vivo in a transfection" to "transfection" (line 86).

28) Please provide city and state affiliation for "Invitrogen" in "The total RNA was extracted from cell lines by the TRIZOL reagent (Invitrogen) and cDNA was synthesized using a cDNA Synthesis kit (Promega, Madison, WI, USA) according to manufacturer’s instructions" (line 90).

29) Please replace "of CD47, (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′)" with "for CD47 (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-93 TAA ACC AAG GCC AGT AG-3′)" (line 92).

30) From "The cDNA was PCR-amplified with the primers of CD47, (forward: 5′-AGA TCC GGT GGT ATG GAT GAG A-3′; reverse: 5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′), HSPA5 (forward: 5’-CTG GCA AGA TGA AGC TCT CC-3’; reverse: 5’-AAA ACC CGA CAG AGG GAC AT-3’), HYOU1 (forward: 5’- GAC TTC GGC ATC TGA GTG GT-3’; reverse: 5’-GCT CCC AAG TCC ACC ATT AC-3’), PDIA4 (forward: 5’- GCT CAG CTC CAG GGA GAG -3’; reverse: 5’- GAT GAT CTC CAC CCA CCT GT-3’), B-actin (forward: 5’-ATG ATA TCG CCG CGC TCG TCG TC-3’; reverse: 5’-CGC TCG GCC GTG GTG GTG AA-3’) was used as the reference gene for normalization" (line 92) is not clear which genes (CD47, HSPA5, HYOU1, PDIA4, B-actin) were used for normalization?

31) "5’-GTC ACA AT-TAA ACC AAG GCC AGT AG-3′" does not seem to be correct primer sequence notation with respect to "AT-TAA" (line 93).

32) Please change "B-actin" to "β-actin" (line 98).

33) "qPCR was analyzed by the ABI-7500 system (Applied Biosystems, Foster City, CA, USA) and the values were calculated using the 2−ΔΔCT method" is not semantically correct with respect to "qPCR was analyzed by" unless the authors are referring to analysis of the qPCR machine itself (line 100).

34) Please provide more specific and technically detailed information on how was RNA-seq quantification performed in the "2.4. RNA-seq quantification" Materials and Methods section.

35) Please provide city name in Taiwan for the affiliation of Illumina in "Differential gene expression was analyzed by RNA-seq quantification following the protocol of Illumina (Genomics, Taiwan), which obtained approximately 10 million reads for each sample" (line 104).

36) Please provide more specific and technically detailed information on how was IPA analysis performed in the "2.5. Ingenuity® Pathway Analysis (IPA)." Materials and Methods section. This needs to be relevant from the experimental point of view.

37) Please replace "Ingenuity® Pathway Analysis (IPA)." with "Ingenuity pathway analysis" (line 111).

38) Please format "Ingenuity® Pathway Analysis (IPA)." using italics (line 111).

39) "IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses" (line 112) sounds vague and rather irrelevant.

40) Please provide city name in USA for the affiliation of Ingenuity Systems in "IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses" (line 112).

41) Please change "CA" to "CA, USA" (lines 112, 128).

42) "Provide data classification and comprehensive explanation" (line 113) does not make sense. Please revise.

43) Please change "In our study, IPA" to "IPA" (line 114).

44) It is not exactly clear what the authors mean by "CD47 downstream candidate genes" in "In our study, IPA was used to conduct network analysis of CD47 downstream candidate genes and explore related signaling pathways information" (line 114)?

45) Please provide more specific and technically detailed information on how was DAVID analysis performed in the "2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis." Materials and Methods section. This needs to be relevant from the experimental point of view.

46) Please replace "(DAVID) analysis." with "analysis" (line 117).

47) Please format "2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis." using italics (line 117).

48) "DAVID analysis is a powerful tool to the function of gene classification" does not seem to make sense (line 119). What is "function of gene classification"?

49) In addition, "DAVID analysis is a powerful tool to the function of gene classification" is not grammatically correct with respect to "tool to the function of" (line 119).

50) "Integrate data from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological processes" (line  119) does not make sense.

51) Similarly, "Calculate the similarity of the global annotation map through agglomeration algorithm and perform integrated classification according to biological functions, signal pathways and types of diseases" (line 121) does not make sense.

52) Please change "analysis." to "analysis" (line 126).

53) Please format "2.7. Statistical analysis." using italics (line 126).

54) Please indent "The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells." on a new line (line 126).

55) "The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells" is not grammatically correct with respect to "expression level were" and "overexpression CD47 plasmid" (line 126). Please rephrase.

56) Please replace "plasmid(1ug, 24hr)" with "plasmid (1 μg, 24 hr)" (line 127).

57) Please change "All of the" to "All" (line 127).

58) Please provide city name in USA for the affiliation of GraphPad Software in "All of the statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Software, CA)" (line 127).

59) "Flowchart showing the gene expression profiling" does not seem to be a meaningful name for section 3.1. Please replace with a title that would reflect on the main finding in this subsection.

60) Please replace "overexpression in oral cancer" with "overexpression" (line 134).

61) From "In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC)" (line 135) is not clear what was the overall proportion of Asian patients enrolled in the study?

62) Please change "Asian who got the" to "Asian patients suffering from" (line 135).

63) Please replace "are" with "is" (lines 136, 138).

64) Please change "betel-quid" to "betel quid" (line 137).

65) Please change "habits" to "habit" (line 139).

66) Please replace "smoking[24,25].Therefore" with "smoking [24,25]. Therefore" (line 139).

67) Please remove "Therefore, that is the reason that we choose the 2 tested cell lines." (line 139).

68) The fact that Illumina NextSeq Sequencing was used is repeatedly stated in "Thus, this study used Illumina NextSeq Sequencing to analyze the genes of OECM-1 and OC-2 and whether they were 141 regulated by CD47" (line 140) and "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142). Please remove this redundancy.

69) Please change "Thus, this study" to "We" (line 140).

70) From "Thus, this study used Illumina NextSeq Sequencing to analyze the genes of OECM-1 and OC-2 and whether they were regulated by CD47" (line 140) is not evident the version of the Illumina NextSeq Sequencing used. Please fix.

71) Please replace "of OECM-1 and OC-2" with "in OECM-1 and OC-2 cells" or "in OECM-1 and OC-2 cell lines" (line 141).

72) "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142) is not a precise statement since Fig. 1A does not only show "that total RNA was extracted from the oral cancer cells". Please rephrase to summarize the overall meaning of Figure 1A.

73) "as per other bioinformatic studies" seems to be a redundant information in "Fig. 1A shows that total RNA was extracted from the oral cancer cells, and Illumina NextSeq Sequencing was applied as per other bioinformatic studies" (line 142). Please remove.

74) Please change "cells," to "cells" (line 142).

75) "In addition, the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B)" (line 143) does not make sense with respect to "the level of CD47 was induced by CD47 plasmid". Do the authors actually mean to say that CD47 was overexpressed?

76) Please replace "OC-2(Fig. 1B)" with "OC-2 cells (Fig. 1B)" or "OC-2 cell lines (Fig. 1B)" (line 145).

77) Please change "OECM-1(CD47/C)" to "OECM-1 (CD47/C)", "OC-2(CD47/C)" to "OC-2 (CD47/C)", "Gene Ontology (GO)" to "Gene ontology (GO)", and "DGE Report" to "DGE report" in Figure 1A.

78) The caption "Derived from Taiwanese patient" seems to clash with the black horizontal arrow in Figure 1A. Please fix.

79) Please indicate statistical significance in Figures 1B, 4E–I.

80) From the legend to Figure 1B is not clear what control was used for qRT-PCR?

81) From the legend to Figure 1B is not clear what is "ClusterProfiler"?

82) Please define abbreviation for "DGE" in the legend to Figure 1A.

83) Please replace "CD47-related" with "CD47" (line 148).

84) Please change "approaches.(B) Overexpress" to "approaches. (B) Overexpression" (line 151).

85) Please replace "transfecting CD47 plasmid(1 ug)" with "transfecting cells with CD47 plasmid (1 μg)" (line 151).

86) Please change "The potential gene expression was identified for CD47-mediated oral cancer" to something like "Gene expression profile in CD47-overexpressing oral cancer" (line 155).

87) Please replace "CD47 overexpression with" with "CD47-overexpressing" (line 158).

88) Please change "(Fig. 2A) in a volcano plot are shown in Fig. 2" to "(Fig. 2A)" (line 158).

89) Please replace "RNA-sequencing heatmap analysis showing genes" with "Genes" (line 159).

90) Please replace "-log10 (p-value)" with "-log10(P value)" (lines 160, 170).

91) Please remove italics formatting from "P" in "p-value" (lines 160, 170).

92) Please change "(Fig. 2B) were chosen for analyses" to "were chosen for further analyses (Fig. 2B)" (line 160).

93) From "Our results identified that the expression of 16 upregulated and 16 downregulated genes was found by CD47 overexpression in OECM-1/OC-2 compared to the control" (line 161) is not clear what control was used to compare gene expression following CD47 overexpression?

94) Please replace "found by" with "found to be associated with", "found to be modulated by" or "found to be regulated by" (line 162).

95) Please change "OECM-1/OC-2" to "OECM-1 and OC-2 cells" or "OECM-1 and OC-2 cell lines" (lines 162, 195). 

96) Please replace "(Fig. 2C). Table 1 lists the 32 association genes." with "(Fig. 2C) (Table 1)" (line 162).

97) Please change "CD47 overexpression on" to "CD47-overexpressing" (lines 166, 168).

98) Please replace "genes, showing that CD47 mediated 617 genes that were upregulated and 653 genes that were downregulated" with "genes show that 617 genes were upregulated and 653 genes were downregulated" (line 171).

99) From Table 1 is not clear what does "M1-C" refer to? Please explain in a legend.

100) From Table 1 is also not clear why is luteolin compared to control?  Please explain the experimental design for luteolin use in a legend. Also, please specify the control.

101) Lastly, from Table 1 is not evident what does "head to head" (SMC2-AS1) and "readthrough" (TRIM6-TRIM34) indicate? Please explain in a legend.

102) Please define abbreviation for "NA" in the legend to Table 1.

103) Please change "changes by the overexpression oof" to "changes induced by the overexpression of" (line 175).

104) Please replace "(DEGs) in CD47-mediated OSCC" with "in CD47-overexpressing oral squamous cell carcinoma" (line 179).

105) Please change "analysis, with a Venn diagram showing the number" to "analysis" (line 181).

106) Please replace "oral cancer (OECM-1/OC-2)" with "OECM-1 and OC-2 oral cancer" (line 184).

107) Please change "(Fig. 3A), Furthermore" to "(Fig. 3A). Furthermore" (line 184).

108) Please replace "demonstrate" with something like "display" or "plot" (line 185).

109) Please change "(Fig. 3B), with Fig. 3C presenting the 14 association genes" to "(Fig. 3B,C)" (line 185).

110) It is not clear what do the numbers in brackets indicate in Figure 3A? Please explain in the respective figure legend.

111) Please flip horizontally Figure 1A so that OECM-1 circle is on the left and OC-2 on the right.

112) Analogously, flip horizontally Figure 1B so that OC-2 control panel is on the left and OC-2-CD47 on the right.

113) The meaning of the heatmap is not clearly defined in Figure 1B. What is the parameter displayed and what does yellow and dark blue color indicate? Please explain to the readers.

114) It is not clear what does the horizontal and vertical cladogram indicate in Figure 3B? Please explain in the respective figure legend.

115) From Figure 3C is not clear what does "NMD candidate" (NPHP3-ACAD11, RTEL1-TNFRSF6B) and "readthrough" (PHOSPHO2-KLHL23, TRIM6-TRIM34) refer to? Also, please define abbreviation for "NMD" in the respective figure legend.

116) Please replace "Gene Symbol" with "Gene symbol", "Official Gene Symbol" with "Official gene symbol", "Gene Name" with "Gene name", "cell lines" with "Cell line", and "OECM-1/ OC-2" with "OECM-1/ OC-2" 7x in Figure 3C.

117) Please replace "function of DEGs on CD47 overexpression in" with "function of differentially expressed genes in CD47-overexpressing" or "predicted function of differentially expressed genes in CD47-overexpressing" (line 190).

118) Please change "comparison" to "comparison was" (line 191).

119) Please replace "upregulated/downregulated genes" with "DEGs" (line 194).

120) Please change "We also" to "In addition, we" (line 197).

121) Please replace "by CD47 overexpression in the" with "CD47-overexpressing" (line 200).

122) Please change "appeared in the MFs, BPs, and CCs (Fig. 4B)" to "appear in all three categories" (line 206).

123) Please replace "on" with "in" (line 207).

124) Please change "use" to "used" (line 208).

125) Please replace "survival ability" with "on the ability of cells to survive" (line 209).

126) Please change "carcinoma[27]" to "carcinoma [27]" (line 210).

127) Please replace "select" with "selected" (line 211).

128) Please change "carcinoma(499 patients sample)" to "carcinoma (499 patients sample)" (line 211).

129) Please format "pression and survival analysis. The results show that the fragments per kilobase per mil-" (line 212) consistently with the rest of the text.

130) Please replace "million(FPKM)" with "million (FPKM)" (line 212).

131) Please change "22.19±10.51, 224.2±87.32, 25.35±13.45, 84.83±37.3" to "22.19 ± 10.51, 224.2 ± 87.32, 25.35 ± 13.45, 84.83 ± 37.3" (line 213).

132) From "The increased expression of HSPA5 (cut off 25%), HYOU1(cut off 75%), and PDIA4(cut off 25%) was significantly associated with survival rate in 499 patient(Fig. 4C)" (line 214) is not clear what the authors mean by "cut off"?

133) Please replace "HYOU1(cut off 75%), and PDIA4(cut off 25%)" with "HYOU1 (cut off 75%), and PDIA4 (cut off 25%)" (line 215).

134) Please change "patient(Fig. 4C)" to "patients (Fig. 4C)" (line 216).

135) From "In addition, HSPA5 was highly expressed in cancer and positively and significantly correlated with CD47 (R=0.099, P = 0.027, Fig. 4D)" (line 216) is clear what does R refer to?

136) Although the authors claim that "In addition, HSPA5 was highly expressed in cancer and positively and significantly correlated with CD47 (R=0.099, P = 0.027, Fig. 4D)" (line 216), the correlation coefficient value is only 0.099 (very weak or no association). Please resolve this dichotomy by acknowledging either the low level of correlation or the absence of it in the text.

137) Please change "R=0.099" to "R = 0.099" (line 217).

138) Please replace "In addition to prove HSPA5 were" with "To prove that HSPA5 was" (line 218).

139) Please change "OECM-1/OC-2" to "OECM-1 and OC-2" (lines 219, 224, 245).

140) Please replace "OECM-1(Fig. 4E) and OC-2(Fig. 4F) OSCC cell lines" with "OECM-1 (Fig. 4E) and OC-2 (Fig. 4F) cell lines" (line 220).

141) Please change "verify" to "verified" (line 221).

142) Please replace "HSPA5" with "HSPA5 overexpression (line 221).

143) Please replace "lines," with "lines." (line 221).

144) Please change "transfer to cell lines and the gene overexpression" to "transfected into the cells and HSPA5 expression" (line 222).

145) Please replace "qRT-PCR(Fig. 4G)" with "qRT-PCR s(Fig. 4G)" (line 223).

146) Please change "The cell" to "Cell" (line 223).

147) Please replace "found" with "revealed" (line 223).

148) From "these data suggests that HSPA5, HYOU1 and PDIA4 was significantly associated with survival rate and HSPA5 was suitable prognostic markers of CD47 mediated in cancer" (line 224) is not clear what the authors mean by "CD47 mediated in cancer" (line 224)?

149) Please change "these data suggests" to "These data suggest" or to something like "Altogether, these data suggest" (line 224).

150) Please replace "PDIA4 was" with "PDIA4 were" (line 225).

151) Please change "HSPA5 was" to "HSPA5 is a" (line 225).

152) Please replace "markers" with "marker" (line 226).

153) Figure 4A is rather pixelated, please enlarge and/or increase its resolution.

154) Please replace "Focus molecular" to "Molecular focus", "SDF2L1/HSPA5/CALR/HYOU1/SESN2/PDIA4/" with "SDF2L1, HSPA5, CALR, HYOU1, SESN2, PDIA4", "CHAC1/CTH/ DDIT3/HERPUD1/HSPA5/SESN2/XBP1/SDF2L1/DERL3/ ASNS/HYOU1/CALR/PDIA4" with "CHAC1, CTH, DDIT3, HERPUD1, HSPA5, SESN2, XBP1, SDF2L1, DERL3, ASNS, HYOU1, CALR, PDIA4", and "HSPA5/SDF2L1/CALR/SLC3A2/SLC1A5/PDIA4/SLC1A4/HYOU1" with "HSPA5, SDF2L1, CALR, SLC3A2, SLC1A5, PDIA4, SLC1A4, HYOU1" in Figure 4A.

155) From Figure 4A is not clear what the authors mean by "Focus molecular"? Please explain in the respective figure legend.

156) Please remove the two small black lines present under the label "B" in Figure 4B.

157) Please remove "Number of values: Sample number" from Figure 4C.

158) Please change "Fragments Per Kilobase of transcript per Million mapped reads (FPKM)" to "Fragments per kilobase of transcript per million mapped reads (FPKM)" in Figure 4C.

159) Please define abbreviation for "CI" under the table in Figure 4C.

160) From the legend to Figure 4C is not clear what does "Number of values" exactly refer to as "Number of values: Sample number" does not provide much clue?

161) Please replace "(B)Three" with "(B) Three" (line 233).

162) Please change "OSCC.(C, D)" to "OSCC. (C, D)" (line 235).

163) "The gene expression and survival rate was analyzed by FPKM of CD47, HSPA5, HYOU1 and PDIA4 through HPA database" (line 235) does not seem to make sense with respect to "gene expression and survival rate was analyzed by FPKM". Please rephrase.

164) Please format "The gene expression and survival rate was analyzed by FPKM of CD47, HSPA5, HYOU1 and PDIA4 through HPA database. (E,F)" (line 235) consistently with the rest of the text.

165) Please replace "OECM-1 and OC-2 cells overexpressed with CD47" with "CD47-overexpressing OECM-1 and OC-2 cells" (line 337).

166) Please change "qRT-237 PCR.(G)HSPA5" to "qRT-237 PCR. (G) HSPA5" (line 237).

167) Please format "gene expression was analyzed by qRT-PCR and the (H, I)cell growth was analyzed by CCK-8 assay." (line 238) consistently with the rest of the text.

168) Please replace "and the (H, I)cell" with "and (H, I) cell" (line 238).

169) From "*P < 0.05 vs. untreated control; two-tailed Student’s t-test" (line 239) is not clear what the authors refer to by "untreated control" as there were not drugs indicated that the cells were treated with?

170) Please format "*P < 239 0.05 vs. untreated control; two-tailed Student’s t-test." (line 239) consistently with the rest of the text.

171) Please change "DEGs in CD47 overexpression" to "differentially expressed genes following CD47 overexpression" or "differentially expressed genes in CD47-overexpressing oral squamous cell carcinoma" (line 242).

172) "We also used DAVID biological process analysis to analyze the function of the DEGs with CD47 overexpression in the OECM-1 and OC-2 cell lines from RNA sequencing" does not seem to make sense with respect to "function of the DEGs with CD47 overexpression in the OECM-1 and OC-2 cell lines from RNA sequencing" (line 243). Please revise.

173) Please replace "with CD47 overexpression in the" with "CD47-overexpressing" (line 244).

174) Please change "separately concluded" to "concluded" (line 245).

175) Please replace "to endoplasmic" with "endoplasmic" (line 247).

176) Please change "to unfolded" to "unfolded" (line 248).

177) Please replace "OECM-1" with "OECM-1 cells" (line 250).

178) Please change "OC-2" with "OC-2 cells" (line 251).

179) Please replace "41genes" with "41 genes" (line 251).

180) Figure 5 is rather pixelated, please enlarge and/or increase its resolution.

181) From the legend to Figure 5 is not clear, what the numbers associated with different processes indicate?

182) Please change "on CD47 overexpression in the" to "in CD47-overexpressing" (line 258).

Response: Following the Reviewer’s comment, we have made the correction accordingly for 182 Minor points in the revision manuscript. 

Round 2

Reviewer 2 Report

The authors have clearly addressed all my concerns associated with this research; therefore, I believe this manuscript is suitable for publication in its present form. 

Author Response

Response: Thanks for reviewer’s comment.

Reviewer 3 Report

I have no further comments.

Author Response

Response: Thanks for reviewer’s comment.

Reviewer 4 Report

Major points:

1) The authors are requested to experimentally address the question whether HSPA5 induces CD47 expression? Please provide the resulting data including statistical significance in the main text of the manuscript or as a supplementary file. This has not been done in the previous round of revision and therefore this point is still pending. Also, please include a short commentary in the discussion section.

2) Please indicate statistical significance of the plots depicted in Figures 4E–I.

Minor points:

1) "A total of 32 top up and -down expression genes were listed, as well as 14 differentially expressed genes were listed" (line 21) is puzzling as to why are 32 top upregulated and downregulated genes mentioned next to 14 differentially expressed genes? Are these also not differentially expressed?

2) Please change "top-up and -down expression" to "top upregulated and downregulated expression" (line 22).

3) Please replace "Pathway Analysis" with "pathway analysis" (line 23).

4) Please replace "to analyzed" with "was used to analyze" (line 25).

5) Please change "was" to "were" (line 27).

6) "which manage to phagocytosis by phagocytes" does not seem to make sense in "Related studies have shown that the expression of CD47 increases in migrating hematopoietic stem cells (HSCs), which manage to phagocytosis by phagocytes" (line 61)? How can HSCs manage to phagocytosis by phagocytes?

7) Please replace "In the Immunotherapy" with "During immunotherapy" (line 68).

8) Please change "receptor(CAR)-T cells that" to "receptor (CAR)-T cells" (line 68).

9) Please replace "killed" with "kill" (line 69).

10) Please change "cell lines" to "cells" (line 70).

11) Please replace "Interesting" with "Interestingly" (line 71).

12) Please change "immunotherapy," to "immunotherapy as" (line 72).

13) Please replace "CD47" with "CD47 was" (line 77).

14) Please change "near" to something like "in proximity to" (line 78).

15) Please replace "have" with "have been" (line 79).

16) It is not exactly clear what the authors mean by "associated through bioinformation" in "In the present study, we examined gene expression profiles by NGS and associated through bioinformation in OSCC cell lines" (line 80)?

17) The sentence "The effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2" (line 81) does not have a verb. Please revise.

18) Please change "(HSPA5, HYOU1, and PDIA4)" to "HSPA5, HYOU1, and PDIA4" (line 82).

19) Please replace "within in" with "within" (line 83).

20) Please change "Gibco, Courtaboeuf, France" to "Gibco" (line 90).

21) Please replace "Brief" with "Briefly" (line 120).

22) Please change "beads" to "were used" (line 120).

23) "Brief, Poly-T oligo-attached beads to purify mRNA and reverse-transcribed synthesis double-strand cDNA, and then sequenced on Illumina NextSeq 500 platform, which obtained approximately 10 million reads for each sample" (line 120) is not grammatically correct with respect to "reverse-transcribed synthesis double-strand cDNA".

24) Please replace "overexpression" with "overexpressing" (line 123).

25) It is not exactly clear what the authors mean by "significantly different genes" in "IPA software (Ingenuity Systems, Redwood City, CA, USA) comprehensively explore significantly different genes to determine their biological function and signaling pathway. The differentially expressed genes were mapped to IPA the genetic network and calculate the score then sorted by score" (line 129)? Do the authors actually mean DEGs?

26) Please change "IPA" to "The IPA" (line 129).

27) Please replace "explore" with "explores" (line 129).

28) Please change "pathway" to "pathways" (line 131).

29) From "The differentially expressed genes were mapped to IPA the genetic network and calculate the score then sorted by score" is not clear what the authors mean by "IPA the genetic network" and "calculate the score then sorted by score"?

30) Please replace "differentially expressed genes" with "DEGs" (lines 131, 206).

31) "IPA was used to conduct network analysis of CD47 downstream mediate genes and explore related signaling pathways information" (line 134) is not grammatically correct with respect to "downstream mediate genes". Moreover, "and explore related signaling pathways information" does not seem to fit the sentence. Please fix. 

32) Please change "differentially expressed genes (DEG)" to "DEGs" (lines 184, 225).

33) Please replace "mediating" with "mediate" (line 357).

34) Please change "expression, our" to "expression. Our" (line 357).

Author Response

Major points:

1) The authors are requested to experimentally address the question whether HSPA5 induces CD47 expression? Please provide the resulting data including statistical significance in the main text of the manuscript or as a supplementary file. This has not been done in the previous round of revision and therefore this point is still pending. Also, please include a short commentary in the discussion section.

Response: Following the Reviewer’s comment, we added new results in Figure 4J and results section “Next, we also analyzed the CD47 expression in HSPA5-overexpressing OSCC cell lines. The results found that CD47 expression was induced by HSPA5-overexpressing in cell lines (Fig. 4J).”, and discussion section “Our data also found that overexpression CD47 induced the expression of HSPA5 and overexpression HSPA5 induced the expression of CD47 in the OECM-1 and OC-2. Previous study also demonstrated that inhibition of glucose-regulated protein-78 (GRP78), a member of HSP70 family, downregulated CD47 expression in tumor cells [46]. Therefore, we believed that CD47 and HSPA5 have a feedbackloop in OSCC cell lines and suggested that CD47 mediated cancer progression through HSPA5 signaling pathway in oral squamous cell carcinoma.”

2) Please indicate statistical significance of the plots depicted in Figures 4E–I.

Response: Following the Reviewer’s comment, we have made the correction in the Figure 4.

Minor points:

1) "A total of 32 top up and -down expression genes were listed, as well as 14 differentially expressed genes were listed" (line 21) is puzzling as to why are 32 top upregulated and downregulated genes mentioned next to 14 differentially expressed genes? Are these also not differentially expressed?

2) Please change "top-up and -down expression" to "top upregulated and downregulated expression" (line 22).

3) Please replace "Pathway Analysis" with "pathway analysis" (line 23).

4) Please replace "to analyzed" with "was used to analyze" (line 25).

5) Please change "was" to "were" (line 27).

6) "which manage to phagocytosis by phagocytes" does not seem to make sense in "Related studies have shown that the expression of CD47 increases in migrating hematopoietic stem cells (HSCs), which manage to phagocytosis by phagocytes" (line 61)? How can HSCs manage to phagocytosis by phagocytes?

7) Please replace "In the Immunotherapy" with "During immunotherapy" (line 68).

8) Please change "receptor(CAR)-T cells that" to "receptor (CAR)-T cells" (line 68).

9) Please replace "killed" with "kill" (line 69).

10) Please change "cell lines" to "cells" (line 70).

11) Please replace "Interesting" with "Interestingly" (line 71).

12) Please change "immunotherapy," to "immunotherapy as" (line 72).

13) Please replace "CD47" with "CD47 was" (line 77).

14) Please change "near" to something like "in proximity to" (line 78).

15) Please replace "have" with "have been" (line 79).

16) It is not exactly clear what the authors mean by "associated through bioinformation" in "In the present study, we examined gene expression profiles by NGS and associated through bioinformation in OSCC cell lines" (line 80)?

17) The sentence "The effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2" (line 81) does not have a verb. Please revise.

18) Please change "(HSPA5, HYOU1, and PDIA4)" to "HSPA5, HYOU1, and PDIA4" (line 82).

19) Please replace "within in" with "within" (line 83).

20) Please change "Gibco, Courtaboeuf, France" to "Gibco" (line 90).

21) Please replace "Brief" with "Briefly" (line 120).

22) Please change "beads" to "were used" (line 120).

23) "Brief, Poly-T oligo-attached beads to purify mRNA and reverse-transcribed synthesis double-strand cDNA, and then sequenced on Illumina NextSeq 500 platform, which obtained approximately 10 million reads for each sample" (line 120) is not grammatically correct with respect to "reverse-transcribed synthesis double-strand cDNA".

24) Please replace "overexpression" with "overexpressing" (line 123).

25) It is not exactly clear what the authors mean by "significantly different genes" in "IPA software (Ingenuity Systems, Redwood City, CA, USA) comprehensively explore significantly different genes to determine their biological function and signaling pathway. The differentially expressed genes were mapped to IPA the genetic network and calculate the score then sorted by score" (line 129)? Do the authors actually mean DEGs?

26) Please change "IPA" to "The IPA" (line 129).

27) Please replace "explore" with "explores" (line 129).

28) Please change "pathway" to "pathways" (line 131).

29) From "The differentially expressed genes were mapped to IPA the genetic network and calculate the score then sorted by score" is not clear what the authors mean by "IPA the genetic network" and "calculate the score then sorted by score"?

30) Please replace "differentially expressed genes" with "DEGs" (lines 131, 206).

31) "IPA was used to conduct network analysis of CD47 downstream mediate genes and explore related signaling pathways information" (line 134) is not grammatically correct with respect to "downstream mediate genes". Moreover, "and explore related signaling pathways information" does not seem to fit the sentence. Please fix. 

32) Please change "differentially expressed genes (DEG)" to "DEGs" (lines 184, 225).

33) Please replace "mediating" with "mediate" (line 357).

34) Please change "expression, our" to "expression. Our" (line 357).

Response: Following the Reviewer’s comment, we have made the correction accordingly for 34 Minor points in the revision manuscript.

Round 3

Reviewer 4 Report

Despite the authors have addressed all major points from the previous round of review, there is still way too many pending corrections that need to be taken care of so that the readers can fully understand and appreciate their study (see below).

1) Please change "C.H" to "C.H." (line 7).

2) Please replace "not so" with "not" (line 18).

3) "In addition, we also used Ingenuity pathway analysis (IPA) was used to analyze the molecular function (MF), biological process (BP), and cellular component (CC) network signaling" (line 22) is not grammatically correct with respect to "used Ingenuity pathway analysis (IPA) was used to".

4) Please change "analyzed" to something like "was used to analyze" (line 24).

5) Please replace "HYOU1 and" with "HYOU1, and" (lines 25, 255, 257).

6) Please replace "was" with "were" (line 26).

7) Please change "were" to "was" (lines 27, 142).

8) Please replace "CD47" with "CD47 expression" (line 27).

9) Please change "bioinformatic" to "bioinformatics" (line 31).

10) "Studies have shown that the occurrence of oral cancer is related to multiple risk factors, including tobacco, drinking, betel chewing, human papillomavirus (HPV) [7], a weakened immune system, a lack of nutrition in the diet, and a poor lifestyle—in particular, betel chewing, excessive alcohol consumption, and smoking cause the increased incidence and mortality of OSCC [8,9]" (line 44) is too long. Please split into two sentences.

11) Please change "factors, including" to "factors including" (line 45)

12) Please replace "receptors, including" with "receptors including" (line 51).

13) It is not exactly clear what the authors mean by "receptors of structural relationships" in "For example, CD47 is the ligand of SIRPα, which is a transmembrane receptor of the immunoglobulin (Ig) superfamily with extracellular immunoglobulin-like domains and antigen receptors of structural relationships" (line 53)?

14) Please change "functions, including" to "functions including" (line 59).

15) Please replace "(HSCs)" with (HSCs), which" (line 61).

16) It is not exactly clear what the authors mean by "high-expressing CD47" in "More evidence indicates that high-expressing CD47 is a common mechanism in human solid tumor cells" (line 64)?

17) Please change "mechanism" to something like "feature" (line 64).

18) "tumor cells"/"tumor cell" is mentioned twice in "Through these mechanisms, these tumor cells can protect themselves from phagocytosis and result in tumor cell proliferation and metastasis" (line 65). Please fix.

19) "and result in tumor cell proliferation and metastasis" does not make sense in "Through these mechanisms, these tumor cells can protect themselves from phagocytosis and result in tumor cell proliferation and metastasis" (line 65) as it is hard to imagine that "tumor cells" can "result in tumor cell proliferation and metastasis". Please revise.

20) Please replace "cells that bind CD47 antigen to" with either "cells that bind CD47 antigen" or "cells bind CD47 antigen to" (line 67).

21) Please change "as, silencing" to "as silencing" (line 70).

22) Please replace "can eliminate" with "eliminated" (line 71).

23) Please change "show" to "have shown" (line 73).

24) Please replace "associated" with something like "associated these data" (line 76).

25) Please change "bioinformation analyzes" to "bioinformatic analyses" (line 77).

26) Please remove "The upregulated and downregulated gene expression in CD47-overexpressing with OSCC cell lines OECM-1 and OC-2" (line 77) as it does not make sense.

27) Please change "CD47 and" to "CD47" (line 80).

28) Please define "empty control" in the Materials and Methods section.

29) It is not clear at which step was the TurboFect transfection mixture added to cells in "2.2. Plasmid transfection"? Please provide this information in the respective section.

30) Please replace "to the" with "to" (line 93).

31) Please replace "TRIZOL" with "TRIzol" (line 96).

32) "Briefly, Poly-T oligo-attached were used to purify mRNA and reverse-transcriptase synthesis double-strand cDNA, and then sequenced on Illumina NextSeq 500 platform, which obtained approximately 10 million reads for each sample" (line 112) seems not to be grammatically correct with respect to "and reverse-transcriptase synthesis double-strand cDNA". Please revise.

33) Please change "in the CD47 overexpressing and control groups in the" to "between the CD47-overexpressing and control" (line 116).

34) Please define abbreviation for "DGE" (line 118), "qRT-PCR" (line 167).

35) Please replace "analysis." with "analysis" (lines 120, 129).

36) Please replace "explore differentially expressed genes (DEGs)" with "explores DEGs" (line 122).

37) Please change "IPA the" to "the IPA" (line 123).

38) "The DEGs were mapped to IPA the genetic network and calculate the score then sorted by score" (line 123) is not grammatically correct with respect to "and calculate the score then sorted by score". Please rephrase.

39) It is not exactly clear what the authors mean by "CD47 downstream mediated genes" in "IPA was used to conduct network analysis of CD47 downstream mediated genes and explores related genetic network" (line 125)?

40) Please replace "explores related genetic network" with something like "to explore the underlying genetic networks" (line 126).

41) "Integrate data and calculate the similarity of the global annotation map from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes" (line 130) is grammatically not correct as it lacks a subject.

42) Please change "databases, including" to "databases including" (line 132).

43) Please replace "(CCK-8) Kit" with "(CCK-8)" (line 136).

44) Please change "1X104" to "1 × 104" (line 137).

45) Please format "4" in "1X104" using superscript (line 137).

46) Please replace "A 10 μL of CCK-8 solution was added into 96-well, and incubated at 37°C" with "Cells were incubated with 10 μL of CCK-8 solution at 37 °C" (line 137).

47) Please replace "37°C" with "37 °C" (line 138).

48) Please change "reader(Thermo, Multiskan GO, USA)" to "reader (Multiskan GO, Thermo Scientific, USA)" (line 139).

49) Please provide city headquarters for "Thermo" in "The OD value was measured at 450 nm with a microplate reader(Thermo, Multiskan GO, USA)" (line 138).

50) From "The gene expression were compared between overexpression CD47 (1 ug, 24 hr) and normal cells" is puzzling why are transfection parameters "(1 ug, 24 hr)" provided as part of the "2.8. Statistical analysis" section? 

51) Please change "overexpression CD47" to "CD47 overexpression" (line 142).

52) Please replace "ug" with "μg" (lines 142, 166).

53) Please replace "Flowchart showing the gene" with "Gene" (line 148).

54) Please change "Most studies" to "Studies" (line 150).

55) Please replace "oral squamous cell carcinoma (OSCC)" with "OSCC" (line 151).

56) Please change "OECM-1, which" to "OECM-1" (line 152).

57) Please replace "OC-2, which" with "OC-2" (line 153).

58) Please change "chewing" to "chewing," (line 155).

59) Please replace "Sequencing" with "sequencing" (line 156).

60) Please change "cells lines" to "cells" or "cell lines" (line 157).

61) Please replace "CD47(Fig. 1A)" with "CD47 (Fig. 1A)" (line 157).

62) Please change "in OSCC cell line" to "in" (line 158).

63) Please replace "profiler" with "profile" (line 159).

64) Please replace "ClusterProfiler" with "clusterProfiler" or "Cluster profile" in Figure 1.

65) Although the authors claim that "The flow chart shows the method of studying various gene expression changes with overexpressed CD47 in OECM-1 and OC-2 cells through gene expression array and the clusterprofiler was analyzed by bioinformatic approaches including gene onltology (GO) and DEGs" (line 162), what is eing shown in Figure 1 is a "DGE report" and not "DEGs". Please fix.

66) Please change "clusterprofiler" to "clusterProfiler" or "cluster profile" (line 164).

67) Please replace "onltology" with "ontology" (line 165).

68) Please change "Overexpress" to "Overexpression" (line 165).

69) Please replace "SE" with "SD" (line 168).

70) Please change "CD47-overexpressing with" to "CD47-overexpressing" (line 173).

71) Please remove bold formatting from "G" in "Genes" (line 174).

72) Please remove bold formatting from "compared to the empty" (line 177).

73) Please replace "CD47-overexpressing on" with "CD47-overexpressing" (lines 180, 182).

74) Please change "gene expression" to "genes" (line 182).

75) Please replace "OC-2 oral cancer" to "OC-2" (line 182).

76) From "Venn diagram of genes show that genes were upregulated and 653 genes were 185 downregulated in oral cancer" is not evident that altered gene expression was due to CD47 overexpression. Please specify this fact as part of this sentence.

77) Please change "show" to "shows" (line 185).

78) Please replace "Table I" with "Table 1" (line 189).

79) Please format "OEC", "/OEC", and "empty control" in "OECM-1-CD47/OECM-1-empty control" using bold in Table 1.

80) Please format "empty control" in "OC2-CD47/OC2- empty control" using bold in Table 1.

81) Please format "O" in "Galactose-3-O-sulfotransferase 2" using italics in Table 1 (GAL3ST2).

82) Please change "DEGs" to "differentially expressed genes" (lines 194, 252).

83) Please replace "cancer" with "cancer cells" or "cancer cell lines" (line 200).

84) Please change "association" to "associated" (lines 211, 247).

85) Please change "DEGs genes" to "DEGs" (line 211).

86) Please replace "CD47-overexpressing" with "CD47 overexpression" (line 214).

87) Please change "analysis, including" to "analysis including" (line 216).

88) It is not exactly clear what the authors mean by "network analysis by CD47 overexpression" in "Then, we focused on the upregulated genes of the network analysis by CD47 overexpression in the OECM-1 and OC-2 cell lines" (line 217)?

89) Please replace "molecular functions (MFs)" with "molecular functions" (line 220).

90) Please change "biological processes (BPs)" to "biological processes" (line 222).

91) Please replace "cellular components (CC)" with "cellular components" (line 223).

92) Please change "database[26]" to "database [26]" (line 226).

93) From "We know that OSCC occupies a high proportion of 90% of head and neck squamous cell carcinoma" (line 227) is not unambiguously clear whether the authors mean to say that OSCC occupies "a high proportion" or "90%" of "head and neck squamous cell carcinoma?

94) Please replace "in the HPA database, we selectd head and neck squamous cell carcinoma (499 patients sample) for gene expression and survival analysis" with "we selected head and neck squamous cell carcinoma (samples from 499 patients) for gene expression and survival analysis in the HPA database" (line 229).

95) From "The increased expression of HSPA5 (cut off 25%), HYOU1 (cut off 75%), and PDIA4 (cut off 25%) was significantly associated with survival rate in 499 patients" (line 232) is not clear what the authors mean by "cut off"?

96) Please change "in" to "in all" (line 234).

97) Please replace "identify the" with "quantify" (line 238).

98) Please change "qRT-PCRs" to "qRT-PCR" (line 242).

99) Please replace "Cell viability assay also" with something like "In addition, cell viability assay" (line 242).

100) Please change "we also analyzed the" to "we analyzed" (line 243).

101) Please replace "HSPA5-overexpressing in" with "HSPA5 overexpression in both" (line 245).

102) It is not clear what the authors mean by "prognostic marker association" in "These data suggest that HSPA5, HYOU1 and PDIA4 were significantly associated with survival rate and HSPA5 is a suitable prognostic marker association with CD47 in cancer" (line 245)?

103) Please change "HYOU1 and" to "HYOU1, and" (line 246).

104) Please replace "survival rate" with "survival" (line 246).

105) Please change "cut off 25% FPKM" to "Cut off 25% FPKM", "cut off Median FPKM" to "Cut off median FPKM", "cut off 25% FPKM" to "Cut off 25% FPKM" in Figure 4C.

106) Please replace "OC-2" with "OC-2 cells" (line 254).

107) "Molecular focus was involved gene in top diseases and function" is not grammatically correct (line 254). Please revise.

108) It is not clear what the authors mean by "beat survival rate" in "Cut off FPKM of 25, 50, 75% to found the beat survival rate, respectively" (line 258)?

109) Please change "(E,F)" to "(E, F)" (line 259).

110) Please replace "SE" with "SD" (line 262).

111) Please change "with CD47-overexpressing in the" to "in CD47-overexpressing" (line 266).

Author Response

Dear Editor,

We would like to submit the revised version of our manuscript entitled “Using next-generation sequencing and bioinformatics methods to predict new genes that may be regulated by CD47 in oral squamous cell carcinoma” (CIMB-1621956) for publication in the Current Issues in Molecular Biology. We thank the reviewers for their constructive comments on the manuscript. In addition, we have incorporated all reviewers’ comments in the revision. The following is a point-by-point response to the reviewers’ concerns.

Reviewer 4

Despite the authors have addressed all major points from the previous round of review, there is still way too many pending corrections that need to be taken care of so that the readers can fully understand and appreciate their study (see below).

1) Please change "C.H" to "C.H." (line 7).

2) Please replace "not so" with "not" (line 18).

3) "In addition, we also used Ingenuity pathway analysis (IPA) was used to analyze the molecular function (MF), biological process (BP), and cellular component (CC) network signaling" (line 22) is not grammatically correct with respect to "used Ingenuity pathway analysis (IPA) was used to".

4) Please change "analyzed" to something like "was used to analyze" (line 24).

5) Please replace "HYOU1 and" with "HYOU1, and" (lines 25, 255, 257).

6) Please replace "was" with "were" (line 26).

7) Please change "were" to "was" (lines 27, 142).

8) Please replace "CD47" with "CD47 expression" (line 27).

9) Please change "bioinformatic" to "bioinformatics" (line 31).

10) "Studies have shown that the occurrence of oral cancer is related to multiple risk factors, including tobacco, drinking, betel chewing, human papillomavirus (HPV) [7], a weakened immune system, a lack of nutrition in the diet, and a poor lifestyle—in particular, betel chewing, excessive alcohol consumption, and smoking cause the increased incidence and mortality of OSCC [8,9]" (line 44) is too long. Please split into two sentences.

11) Please change "factors, including" to "factors including" (line 45)

12) Please replace "receptors, including" with "receptors including" (line 51).

13) It is not exactly clear what the authors mean by "receptors of structural relationships" in "For example, CD47 is the ligand of SIRPα, which is a transmembrane receptor of the immunoglobulin (Ig) superfamily with extracellular immunoglobulin-like domains and antigen receptors of structural relationships" (line 53)?

14) Please change "functions, including" to "functions including" (line 59).

15) Please replace "(HSCs)" with (HSCs), which" (line 61).

16) It is not exactly clear what the authors mean by "high-expressing CD47" in "More evidence indicates that high-expressing CD47 is a common mechanism in human solid tumor cells" (line 64)?

17) Please change "mechanism" to something like "feature" (line 64).

18) "tumor cells"/"tumor cell" is mentioned twice in "Through these mechanisms, these tumor cells can protect themselves from phagocytosis and result in tumor cell proliferation and metastasis" (line 65). Please fix.

19) "and result in tumor cell proliferation and metastasis" does not make sense in "Through these mechanisms, these tumor cells can protect themselves from phagocytosis and result in tumor cell proliferation and metastasis" (line 65) as it is hard to imagine that "tumor cells" can "result in tumor cell proliferation and metastasis". Please revise.

20) Please replace "cells that bind CD47 antigen to" with either "cells that bind CD47 antigen" or "cells bind CD47 antigen to" (line 67).

21) Please change "as, silencing" to "as silencing" (line 70).

22) Please replace "can eliminate" with "eliminated" (line 71).

23) Please change "show" to "have shown" (line 73).

24) Please replace "associated" with something like "associated these data" (line 76).

25) Please change "bioinformation analyzes" to "bioinformatic analyses" (line 77).

26) Please remove "The upregulated and downregulated gene expression in CD47-overexpressing with OSCC cell lines OECM-1 and OC-2" (line 77) as it does not make sense.

27) Please change "CD47 and" to "CD47" (line 80).

28) Please define "empty control" in the Materials and Methods section.

29) It is not clear at which step was the TurboFect transfection mixture added to cells in "2.2. Plasmid transfection"? Please provide this information in the respective section.

30) Please replace "to the" with "to" (line 93).

31) Please replace "TRIZOL" with "TRIzol" (line 96).

32) "Briefly, Poly-T oligo-attached were used to purify mRNA and reverse-transcriptase synthesis double-strand cDNA, and then sequenced on Illumina NextSeq 500 platform, which obtained approximately 10 million reads for each sample" (line 112) seems not to be grammatically correct with respect to "and reverse-transcriptase synthesis double-strand cDNA". Please revise.

33) Please change "in the CD47 overexpressing and control groups in the" to "between the CD47-overexpressing and control" (line 116).

34) Please define abbreviation for "DGE" (line 118), "qRT-PCR" (line 167).

35) Please replace "analysis." with "analysis" (lines 120, 129).

36) Please replace "explore differentially expressed genes (DEGs)" with "explores DEGs" (line 122).

37) Please change "IPA the" to "the IPA" (line 123).

38) "The DEGs were mapped to IPA the genetic network and calculate the score then sorted by score" (line 123) is not grammatically correct with respect to "and calculate the score then sorted by score". Please rephrase.

39) It is not exactly clear what the authors mean by "CD47 downstream mediated genes" in "IPA was used to conduct network analysis of CD47 downstream mediated genes and explores related genetic network" (line 125)?

40) Please replace "explores related genetic network" with something like "to explore the underlying genetic networks" (line 126).

41) "Integrate data and calculate the similarity of the global annotation map from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes" (line 130) is grammatically not correct as it lacks a subject.

42) Please change "databases, including" to "databases including" (line 132).

43) Please replace "(CCK-8) Kit" with "(CCK-8)" (line 136).

44) Please change "1X104" to "1 × 104" (line 137).

45) Please format "4" in "1X104" using superscript (line 137).

46) Please replace "A 10 μL of CCK-8 solution was added into 96-well, and incubated at 37°C" with "Cells were incubated with 10 μL of CCK-8 solution at 37 °C" (line 137).

47) Please replace "37°C" with "37 °C" (line 138).

48) Please change "reader(Thermo, Multiskan GO, USA)" to "reader (Multiskan GO, Thermo Scientific, USA)" (line 139).

49) Please provide city headquarters for "Thermo" in "The OD value was measured at 450 nm with a microplate reader(Thermo, Multiskan GO, USA)" (line 138).

50) From "The gene expression were compared between overexpression CD47 (1 ug, 24 hr) and normal cells" is puzzling why are transfection parameters "(1 ug, 24 hr)" provided as part of the "2.8. Statistical analysis" section? 

51) Please change "overexpression CD47" to "CD47 overexpression" (line 142).

52) Please replace "ug" with "μg" (lines 142, 166).

53) Please replace "Flowchart showing the gene" with "Gene" (line 148).

54) Please change "Most studies" to "Studies" (line 150).

55) Please replace "oral squamous cell carcinoma (OSCC)" with "OSCC" (line 151).

56) Please change "OECM-1, which" to "OECM-1" (line 152).

57) Please replace "OC-2, which" with "OC-2" (line 153).

58) Please change "chewing" to "chewing," (line 155).

59) Please replace "Sequencing" with "sequencing" (line 156).

60) Please change "cells lines" to "cells" or "cell lines" (line 157).

61) Please replace "CD47(Fig. 1A)" with "CD47 (Fig. 1A)" (line 157).

62) Please change "in OSCC cell line" to "in" (line 158).

63) Please replace "profiler" with "profile" (line 159).

64) Please replace "ClusterProfiler" with "clusterProfiler" or "Cluster profile" in Figure 1.

65) Although the authors claim that "The flow chart shows the method of studying various gene expression changes with overexpressed CD47 in OECM-1 and OC-2 cells through gene expression array and the clusterprofiler was analyzed by bioinformatic approaches including gene onltology (GO) and DEGs" (line 162), what is eing shown in Figure 1 is a "DGE report" and not "DEGs". Please fix.

66) Please change "clusterprofiler" to "clusterProfiler" or "cluster profile" (line 164).

67) Please replace "onltology" with "ontology" (line 165).

68) Please change "Overexpress" to "Overexpression" (line 165).

69) Please replace "SE" with "SD" (line 168).

70) Please change "CD47-overexpressing with" to "CD47-overexpressing" (line 173).

71) Please remove bold formatting from "G" in "Genes" (line 174).

72) Please remove bold formatting from "compared to the empty" (line 177).

73) Please replace "CD47-overexpressing on" with "CD47-overexpressing" (lines 180, 182).

74) Please change "gene expression" to "genes" (line 182).

75) Please replace "OC-2 oral cancer" to "OC-2" (line 182).

76) From "Venn diagram of genes show that genes were upregulated and 653 genes were 185 downregulated in oral cancer" is not evident that altered gene expression was due to CD47 overexpression. Please specify this fact as part of this sentence.

77) Please change "show" to "shows" (line 185).

78) Please replace "Table I" with "Table 1" (line 189).

79) Please format "OEC", "/OEC", and "empty control" in "OECM-1-CD47/OECM-1-empty control" using bold in Table 1.

80) Please format "empty control" in "OC2-CD47/OC2- empty control" using bold in Table 1.

81) Please format "O" in "Galactose-3-O-sulfotransferase 2" using italics in Table 1 (GAL3ST2).

82) Please change "DEGs" to "differentially expressed genes" (lines 194, 252).

83) Please replace "cancer" with "cancer cells" or "cancer cell lines" (line 200).

84) Please change "association" to "associated" (lines 211, 247).

85) Please change "DEGs genes" to "DEGs" (line 211).

86) Please replace "CD47-overexpressing" with "CD47 overexpression" (line 214).

87) Please change "analysis, including" to "analysis including" (line 216).

88) It is not exactly clear what the authors mean by "network analysis by CD47 overexpression" in "Then, we focused on the upregulated genes of the network analysis by CD47 overexpression in the OECM-1 and OC-2 cell lines" (line 217)?

89) Please replace "molecular functions (MFs)" with "molecular functions" (line 220).

90) Please change "biological processes (BPs)" to "biological processes" (line 222).

91) Please replace "cellular components (CC)" with "cellular components" (line 223).

92) Please change "database[26]" to "database [26]" (line 226).

93) From "We know that OSCC occupies a high proportion of 90% of head and neck squamous cell carcinoma" (line 227) is not unambiguously clear whether the authors mean to say that OSCC occupies "a high proportion" or "90%" of "head and neck squamous cell carcinoma?

94) Please replace "in the HPA database, we selectd head and neck squamous cell carcinoma (499 patients sample) for gene expression and survival analysis" with "we selected head and neck squamous cell carcinoma (samples from 499 patients) for gene expression and survival analysis in the HPA database" (line 229).

95) From "The increased expression of HSPA5 (cut off 25%), HYOU1 (cut off 75%), and PDIA4 (cut off 25%) was significantly associated with survival rate in 499 patients" (line 232) is not clear what the authors mean by "cut off"?

96) Please change "in" to "in all" (line 234).

97) Please replace "identify the" with "quantify" (line 238).

98) Please change "qRT-PCRs" to "qRT-PCR" (line 242).

99) Please replace "Cell viability assay also" with something like "In addition, cell viability assay" (line 242).

100) Please change "we also analyzed the" to "we analyzed" (line 243).

101) Please replace "HSPA5-overexpressing in" with "HSPA5 overexpression in both" (line 245).

102) It is not clear what the authors mean by "prognostic marker association" in "These data suggest that HSPA5, HYOU1 and PDIA4 were significantly associated with survival rate and HSPA5 is a suitable prognostic marker association with CD47 in cancer" (line 245)?

103) Please change "HYOU1 and" to "HYOU1, and" (line 246).

104) Please replace "survival rate" with "survival" (line 246).

105) Please change "cut off 25% FPKM" to "Cut off 25% FPKM", "cut off Median FPKM" to "Cut off median FPKM", "cut off 25% FPKM" to "Cut off 25% FPKM" in Figure 4C.

106) Please replace "OC-2" with "OC-2 cells" (line 254).

107) "Molecular focus was involved gene in top diseases and function" is not grammatically correct (line 254). Please revise.

108) It is not clear what the authors mean by "beat survival rate" in "Cut off FPKM of 25, 50, 75% to found the beat survival rate, respectively" (line 258)?

109) Please change "(E,F)" to "(E, F)" (line 259).

110) Please replace "SE" with "SD" (line 262).

111) Please change "with CD47-overexpressing in the" to "in CD47-overexpressing" (line 266).

Response: Following the Reviewer’s comment, we have made the correction accordingly for 111 Minor points in the revision manuscript.

 

We believe we have responded in a positive manner to all Reviewers’ comments, and hope that this would convince the Reviewers that the revision has been significantly improved in its scientific merit for the broad readership of the Current Issues in Molecular Biology Journal.

 

Thank you for your consideration.

 

Sincerely yours,

 

Tsung-Hua Hsieh, PhD

Department of Medical Research, E-Da Hospital/ E-Da Cancer Hospital E-mail address:[email protected]

Mailing address: 6 Yi-Da Rd., Yan-Chao District, Kaohsiung 82445, Taiwan Phone: 886-7-6151100#5072

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript by Tseng and colleagues reports the use of RNA-Seq to identify genes and/or molecular pathways that may be regulated by CD47 in cell lines of oral squamous cell carcinoma.

Oral cell carcinoma is increasing both its incidence and mortality; moreover, it is often diagnosed in advanced stage, thus negatively impacting the overall patients' outcomes and survival. As a consequence, identify molecular biomarkers and pathways that may be used to improve the diagnostic strategies and/or to enhance the development of novel therapies, is an actual issue. In this context, investigate the effects of CD47 over-expression on mRNA profiling may be of interest. However, the study design need to be improved.

First, it is not clear why the 2 tested cell lines were chosen. Are these the only 2 models of OSCC? What are the difference between them? Are they currently used as OSCC in vitro models?

These aspects should be explained in the Introduction section. Moreover, the aims of the study, what is already known about CD47 expression in OSCCs and what issues authors plan to assess have to be clearly stated at the end of the Introduction. 

 

Again, regarding the used cell lines and the methods used herein, what about their basal expression of CD47? The authors  transfected both of them in order to over-express CD47: not only they did not verified the efficiency of the performed transfection, but did not report CD47 basal level or the effect of endogenous CD47 silencing on gene expression. The methods used to carry out the RNA-Seq must be detailed better and also the bioinformatic tools used for data analyses and pathways prediction, and statistic tools need to be described under Methods.

Section 3.1 has to be moved under Methods since it is just the description of the study design and not a result. The study design itself needs to be improved as specified above. Figure 1 may be useful for readers but its really too basic in its actual form.

The other sections of Results have to be improved because are confused and difficult to follow.

The discussion also should point on the potential translational values of the obtained results that does not clearly emerge.

Minor Points:

Check the term "bioinformatics" throughout the text: if used as an adjective it must to be used in the singular (including the title).

After first mention within the text use just the abbreviation

Author Response

Reviewer #1: The manuscript by Tseng and colleagues reports the use of RNA-Seq to identify genes and/or molecular pathways that may be regulated by CD47 in cell lines of oral squamous cell carcinoma.

Oral cell carcinoma is increasing both its incidence and mortality; moreover, it is often diagnosed in advanced stage, thus negatively impacting the overall patients' outcomes and survival. As a consequence, identify molecular biomarkers and pathways that may be used to improve the diagnostic strategies and/or to enhance the development of novel therapies, is an actual issue. In this context, investigate the effects of CD47 over-expression on mRNA profiling may be of interest. However, the study design need to be improved.

First, it is not clear why the 2 tested cell lines were chosen. Are these the only 2 models of OSCC? What are the difference between them? Are they currently used as OSCC in vitromodels?

Response: The Reviewer’s observation is correct, In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC). OECM-1, which are derived from surgical resection of a primary tumor from a Taiwanese patient with a history of betel-quid chewing. OC-2, which are derived from a primary tumor of the buccal mucosa of a Chinese patient with a habits of alcohol drinking, betel quid chewing and cigarette smoking[24, 25]. Therefore, that is the reason that we choose the 2 tested cell lines.

These aspects should be explained in the Introduction section. Moreover, the aims of the study, what is already known about CD47 expression in OSCCs and what issues authors plan to assess have to be clearly stated at the end of the Introduction.

Response: Following the Reviewer’s comment, we added the section in the introduction and end of the introduction.” In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23].” and “In the present study, we examine the effects of CD47 gene expression in  OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4) and lipid drop-associated proteins (HILPDA and PLIN2)were induced by CD47 and involved in the OSCC network.” 

Again, regarding the used cell lines and the methods used herein, what about their basal expression of CD47? The authors  transfected both of them in order to over-express CD47: not only they did not verified the efficiency of the performed transfection, but did not report CD47 basal level or the effect of endogenous CD47 silencing on gene expression. The methods used to carry out the RNA-Seq must be detailed better and also the bioinformatic tools used for data analyses and pathways prediction, and statistic tools need to be described under Methods.

Response: Following the Reviewer’s comment, we added the new results in the Fig. 1B. Overexpress of CD47 was performed by transfecting CD47 plasmid(1 ug) and CD47 expression was detected with qRT-PCR and compared to control. The results found that the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B). In addition, we know that silence CD47 affects downstream regulatory genes is very important, but the exploration of the mechanism is very important and complicated. Therefore, we will focus on the regulation of silence CD47 on downstream genes in more depth in the future. We also added the new bioinformatic tools in Methods. “2.5. Ingenuity® Pathway Analysis (IPA). IPA software (Ingenuity Systems, CA) integrates large amounts of research data and performs multiple analyses. Provide data classification and comprehensive explanation. In our study, IPA was used to conduct network analysis of CD47 downstream candidate genes and explore related signaling pathways information.” , “2.6. Database for annotation, visualization and integrated discovery (DAVID) analysis. DAVID analysis is a powerful tool to the function of gene classification. Integrate data from multiple functional annotation databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological processes. Calculate the similarity of the global annotation map through agglomeration algorithm and perform integrated classification according to biological functions, signal pathways and types of diseases.” and “ 2.7. Statistical analysis. The gene expression level were compared between overexpression CD47 plasmid(1ug, 24hr) and normal cells. All of the statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Software, CA).”

Section 3.1 has to be moved under Methods since it is just the description of the study design and not a result. The study design itself needs to be improved as specified above. Figure 1 may be useful for readers but its really too basic in its actual form.

Response: The Reviewer’s observation is correct, we modification the Fig 1A and added the new results in the Fig. 1B. Overexpress of CD47 was performed by transfecting CD47 plasmid(1 ug) and CD47 expression was detected with qRT-PCR and compared to control. The results found that the level of CD47 was induced by CD47 plasmid in OSCC cell line OECM-1 and OC-2(Fig. 1B).

The other sections of Results have to be improved because are confused and difficult to follow.

Response: Following the Reviewer’s comment, we reversed Figure 4 and Figure 5 as well as adjust the order of Fig 4 A, B and C, and make the manuscript more easier to follow.

The discussion also should point on the potential translational values of the obtained results that does not clearly emerge.

Response: Following the Reviewer’s comment, the potential translational values of the obtained results were show in conclusions section. “These results provide important insights into possible new diagnostic genes for OSCC-targeting CD47. Although the mechanism by which CD47 regulates unfolded proteins and lipid drop-associated proteins is still unclear, the detailed signaling pathway will be discussed in more depth in the future.”

Minor Points:

Check the term "bioinformatics" throughout the text: if used as an adjective it must to be used in the singular (including the title).

After first mention within the text use just the abbreviation

Response: Following the Reviewer’s comment, we have made the correction accordingly.

Reviewer 2 Report

General comments:

This study provides important insights into possible new diagnostic strategies, including unfolded protein and lipid drop-associated protein for OSCC-targeting CD47.

 

Major comments:

1. The aims of this study was not mentioned in the end of the introduction. Please mention about it.
2. The introduction for CD47 was mentioned for a number of cancer but not for oral cancer. It should make a connection to oral cancer cells and becomes the aim of this study.
3. Moreover, some studies for investigating CD47 to oral cancer was not mentioned in oral cancer cells. For example,

(1) Oncol Lett 2018 Jun;15(6):9075-9080 (CD47 as a potential prognostic marker for oral leukoplakia and OSCC).

(2) Cells 2019, 8(12), 1658. (CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma)

Author Response

Reviewer #2: This study provides important insights into possible new diagnostic strategies, including unfolded protein and lipid drop-associated protein for OSCC-targeting CD47.

Major comments:

1. The aims of this study was not mentioned in the end of the introduction. Please mention about it.

Response: Following the Reviewer’s comment, we added the section in the end of the introduction. “In the present study, we examine the effects of CD47 gene expression in OSCC cell lines OECM-1 and OC-2 as well as found that unfolded proteins (HSPA5, HYOU1, and PDIA4) and lipid drop-associated proteins (HILPDA and PLIN2)were induced by CD47 and involved in the OSCC network.”  

2. The introduction for CD47 was mentioned for a number of cancer but not for oral cancer. It should make a connection to oral cancer cells and becomes the aim of this study.

Response: Following the Reviewer’s comment, we added the new section and reference in the introduction. “ In addition, previous studies have found that high expression CD47 was detected in OSCC cell lines and significantly higher in OSCC than near tissues. Furthermore, the poor prognosis in patients with OSCC have connected to CD47-SIRPα signaling pathway[23].”

 

1. Moreover, some studies for investigating CD47 to oral cancer was not mentioned in oral cancer cells. For example,

(1) Oncol Lett 2018 Jun;15(6):9075-9080 (CD47 as a potential prognostic marker for oral leukoplakia and OSCC).

(2) Cells 2019, 8(12), 1658. (CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma)

Response: The Reviewer’s observation is correct, In this study, we mainly focus on Asian who got the oral squamous cell carcinoma (OSCC). OECM-1, which are derived from surgical resection of a primary tumor from a Taiwanese patient with a history of betel-quid chewing. OC-2, which are derived from a primary tumor of the buccal mucosa of a Chinese patient with a habits of alcohol drinking, betel quid chewing and cigarette smoking[24, 25]. Therefore, that is the reason that we choose the 2 tested cell lines.

Reviewer 3 Report

In this manuscript, the authors assay gene changes compared between oral squamous cell carcinoma (OSCC) cell and CD47 transfected OSCC cell.

I think the authors need to demonstrate in clinical data whether the genes they featured are associated with prognosis. Otherwise, most researcher will not be interested in the result.

Author Response

Reviewer #3: In this manuscript, the authors assay gene changes compared between oral squamous cell carcinoma (OSCC) cell and CD47 transfected OSCC cell.

I think the authors need to demonstrate in clinical data whether the genes they featured are associated with prognosis. Otherwise, most researcher will not be interested in the result.

Response: The Reviewer’s observation is correct, we very agree with the comment of esteemed reviewer, and we are also in progress. At this stage, we continue to collect clinical sample, but it will take a long time to receive clinical cases, we cannot get relevant information quickly. We will discuss the any comments by reviewer in our studies in future.

Round 2

Reviewer 1 Report

The authors take into account the previous comments and used them to revise the manuscript that appears improved with respect to the original version.

However, the study design seems to be still confounding and both methods and results sections have to be improved before the manuscript can be considered for publication.

 

Reviewer 3 Report

1. Previous research have demonstrated that CD47-SIRPalpha signaling is associated with poor prognosis(ref no.23).
2. There are no over in vivo experiments

 

I think this manuscript have less novelty from above reasons.

In my opinion, the authors should resubmit the manuscript to another journal with a lower impact factor.