MicroRNA-22-3p and MicroRNA-149-5p Inhibit Human Hepatocellular Carcinoma Cell Growth and Metastasis Properties by Regulating Methylenetetrahydrofolate Reductase

Round 1

Reviewer 1 Report

Li et al. investigated the potential role of miR-22-3p and miR-149-5p in HCC cell lines. However, the data presented in this manuscript do not add any novelty regarding the tumor suppressor functions of the selected miRNAs when compared to the previous studies on human HCC . In addition, the conclusions reported in this study are barely supported by the experimental observations included in the results. Please find below some concerns arose from reviewing this manuscript:

1. Figure 1: why only two HCC cell lines were screened for miRNA expression?
2. Figure 2 legend: what do the letters used over the histogram bars indicate?
3. Figure 3g: miR-149-5p was not used to determine MTHFR mRNA levels, do the authors have an explanation?
4. Results 3.7: Which was the criteria used to select TP53INP1 and PDCD4?
5. As stated in the discussion but without experimental data included in the manuscript (line 266), does MTHFR regulation affect PDCD4 and TP53INP1 in HCC cell lines?
6. Are PDCD4 and TP53INP1 involved in the regulation of proliferation and metastasis of HCC cells? The results included in this manuscript do not support this conclusion (line 268)
7. The results do not demonstrate that miR-22 and miR-149 can inhibit cell proliferation and metastasis by downregulating MTHFR expression, no rescue experiments are included in the study.
8. Line 273: since these results are not shown, they cannot be used to support a key conclusion of the study.
9. Line 282: there are no evidences that MTHFR affects TP53INP1 and PDCD4 in this study. Or has it been demonstrated in other studies?
10. In general, there is no direct evidence that both miR-22-3p and miR-149-5p target MTHFR; the effect seen in Figure 3 might be an indirect effect. Luciferase assay experiments would have been helpful to confirm the in silico prediction as reported in the conclusions.
11. Line 63: reference missed
12. Line 173: reference missed

Author Response

Thanks to the reviewers for their comments, we have revised the article based on the comments and wrote the following responses.

About the conclusion:

We have revised the conclusion to: miRNA-22-3p and miRNA-149-5p inhibit tumor growth and metastasis properties may be partially by targeting MTHFR and that they exert anticancer effects in hepatocellular carcinoma cells.

About details:

1. Figure 1: why only two HCC cell lines were screened for miRNA expression?

Figure 1 shows the endogenous transcription of miR-22-3p/miR-149-5p in two human HCC cell lines (QGY-7703, HepG2) and one normal HL-7702 liver cells selected in the study to provide a control for subsequent experiments. 

2. Figure 2 legend: what do the letters used over the histogram bars indicate?

Because there are three bar graphs in Figure 2, one letter represents a bar graph, showing the proliferation status of a cell line. This can be clearly stated in the description to avoid confusion.  Different letters indicate a significant difference in the histogram bars.

3. Figure 3g: miR-149-5p was not used to determine MTHFR mRNA levels, do the authors have an explanation?

Because of the instability of RNA, the transcription level of MTHFR after transfection with miR-149-5p was not detected. But the protein level results were ideal and sufficiently supportive, so we did not have the results of RNA supplementation.

4. Results 3.7: Which was the criteria used to select TP53INP1 and PDCD4?

Because the main purpose of the article is to study the mechanism of the anti-cancer activity of two small RNAs, TP53INP1 and PDCD4 are recognized tumor suppressor gene markers, so these two genes were selected for the study to verify whether transfection of miR-22-3p/miR-149-5p could alter the two tumor suppressors gene expression.

     5. As stated in the discussion but without experimental data included in the manuscript (line 266), does MTHFR regulation affect PDCD4 and TP53INP1 in HCC cell lines?

Modified in lines 272-273.

    6. Are PDCD4 and TP53INP1 involved in the regulation of proliferation and metastasis of HCC cells? The results included in this manuscript do not support this conclusion (line 268)

Modified in lines 238 and 272-273.

   7. The results do not demonstrate that miR-22 and miR-149 can inhibit cell proliferation and metastasis by downregulating MTHFR expression, no rescue experiments are included in the study.

Results 3.2. MiR-22-3p/miR-149-5p inhibit hepatocellular carcinoma cells proliferation, figure 1 

Results 3.4. Transfection of miR-22-3p/miR-149-5p mimic suppresses migration and invasion in HCC cells in vitro, figure 4

Results 3.5. Transfection of miR-22-3p/miR-149-5p inhibitor promote migration in HCC cells in vitro, figure 5

Results 3.6. Silencing MTHFR inhibit migration in HCC cell lines, figure 5

Based on the above results, it can be confirmed that miR-22-3p and miR-149-5p inhibit the migration and that the invasion properties of HCC may be partially able to be achieved through downregulating MTHFR. Modified in lines 231-233.

    8. Line 273: since these results are not shown, they cannot be used to support a key conclusion of the study.

It has been modified.

    9. Line 282: there are no evidences that MTHFR affects TP53INP1 and PDCD4 in this study. Or has it been demonstrated in other studies?

We have marked in the text: Data not show here.

    10. In general, there is no direct evidence that both miR-22-3p and miR-149-5p target MTHFR; the effect seen in Figure 3 might be an indirect effect. Luciferase assay experiments would have been helpful to confirm the in silicoprediction as reported in the conclusions.

We have verified it in our previous work, and it is also stated in the intrudution. The resulting image is displayed in the attached.

About language:

Manuscript has undergone English language editing by MDPI.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors added new experiments in the revised manuscript, thus improving the quality of the work. However, some of my concerns have not been addressed.

- Based on my previous criticism, the new title is still incorrect. I suggest to change “cells growth and metastasis” with “cell proliferation and metastatic properties” or with “cell proliferation, migration and invasion”. The sentences in line 257 and lines 289-290 should be changed as well.

- The sentence “Reduced MTHFR activities are associated with a high-risk for the development of HCC and are correlated with lower risks for late-stage HCC and a favorable survival of patients with HCC” (lines 60-62) has not been clarified and supported by literature data (ref #8 and #9 are not concerning HCC and ref #10 refers to a specific MTHFR gene polymorphism). The authors should better and more clearly describe the role of MTHFR in HCC.

- The p value corresponding to letters “a”, “b” and “c” in figure 2 has not been indicated.

- The number of independent experiments of Western Blot carried out and utilized for statistical analyses has not been reported. It is not clear whether the graphs of figures 3c, 3f, 3i and 6b, 6d represent the quantification of the gel shown in the same figure or of WB gels from different experiments.

- As previously highlighted, the hypothetical role of the observed TP53INP1 and PDCD4 upregulation by miRNAs in HCC can be ascribed to the promotion of “tumor suppression” rather than to the promotion of “HCC cell apoptosis” (no evidence of apoptosis has been shown in this manuscript).

- English grammar errors and typos are still present in the manuscript.

Finally, the authors should not utilize the same sentences (lines 45-49 and 51-62) already reported in their previously published papers (Li et al., 2017).

Author Response

Comments given by reviewers regarding language, topics, conclusions, and figures have been revised in the manuscript. Include line 3, line 30, line 298, line 305, figure 2, figure 3.

A little explanation:

Reviewers propose: the sentence “Reduced MTHFR activities are associated with a high-risk for the development of HCC and are correlated with lower risks for late-stage HCC and a favorable survival of patients with HCC” (lines 60-62) has not been clarified and supported by literature data (ref #8 and #9 are not concerning HCC and ref #10 refers to a specific MTHFR gene polymorphism). The authors should better and more clearly describe the role of MTHFR in HCC.

Ref#10 mentioned that MTHFR C677T affects the activity of MTHFR and thus affects the survival of HCC patients. This is clearly mentioned in the literature. It means that SNP leads to a decrease in activity, while our manuscript reduces the expression of MTHFR through miRNA. The effects of the two may be similar, we just want to highlight this point, these several papers were applied. The abstract of the ref#10 is provided below (in the attached).

About language:

Manuscript has undergone English language editing by MDPI.

Author Response File: Author Response.pdf

Reviewer 3 Report

Li and colleagues investigate the function of microRNA-22-3p and microRNA-149-5p in hepatocellular carcinoma. Using microRNA mimics and inhibitors authors show the function of these 2 microRNA in cell proliferation, cell migration and cell invasion in vitro. They further demonstrate that modulating MTHFR recapitulate the phenotype indicating that it mimics microRNA functions in vitro. Importantly, these regulations and phenotypical differences are observed in two HCC cell lines, but not in normal liver cells.

 

There are a few typos here and there but overall the manuscript is well written and data clearly explained. I would nonetheless recommend to format all the figures using the same standards (p-value are indicated by letters or asterisks depending on figures…).

 

Main point :

• Authors mention that this study is based on previously published work demonstrating that microRNA-22-3p and microRNA-149-5p regulate MTHFR expression. This study is not cited in the manuscript and I could not find it. It would be best to indicate clearly the reference to this work which is important to interpret the data in here.
• Authors show that microRNAs altered cell proliferation which may impact cell migration and cell invasion assays, notably because these assays required longer cell culture (based on the M&M). It would be best to perform such assays in condition were proliferation is blocked (i.e. hydroxyurea treatment)
• While silencing of MTHFR mimics microRNA phenotypes it is not possible to conclude that « microRNAs inhibit migration of HCC through downregulating MTHFR » (lane 220-221). Such conclusion could only be achieved by re-introducing a microRNA-resistant MTHFR in a microRNA mimic experiment. The conclusion is too strong.
• I do not understand the point of Figure 7. Are PDCD4 and TP53INP1 targets of MTHFR or the microRNAs ?
• Why are the data for microRNA-149-5p not shown in panel g of figure 3 ?

Author Response

Thanks to the reviewers for their comments, we have revised the manuscript based on the comments and wrote the following responses.

     1. Authors mention that this study is based on previously published work demonstrating that microRNA-22-3p and microRNA-149-5p regulate MTHFR expression. This study is not cited in the manuscript and I could not find it. It would be best to indicate clearly the reference to this work which is important to interpret the data in here.

We have marked reference 11 in line 66.

2. The conclusion is too strong.

We have modified the result to :“Based on the above results, it can be confirmed that miR-22-3p and miR-149-5p inhibit the migration and that the invasion properties of HCC may be partially able to be achieved through downregulating MTHFR.” Corresponding changes have also been made elsewhere in the manuscrip.

   3. I do not understand the point of Figure 7. Are PDCD4 and TP53INP1 targets of MTHFR or the microRNAs?

Because the main purpose of the article is to study the mechanism of the anti-cancer activity of two small RNAs, TP53INP1 and PDCD4 are recognized tumor suppressor gene markers, so these two genes were selected for the study to verify whether transfection of miR-22-3p/miR-149-5p could alter the two tumor suppressors gene expression.

4. Why are the data for microRNA-149-5p not shown in panel g of figure 3?

Because of the instability of RNA, the transcription level of MTHFR after transfection with miR-149-5p was not detected. But the protein level results were ideal and sufficiently supportive, so we did not have the results of RNA supplementation.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Reply-Reviewer 1:

The authors have improved the key conclusions of the manuscript. However, there are still some changes needed.

 

About the conclusion:

We have revised the conclusion to: miRNA-22-3p and miRNA-149-5p inhibit tumor growth and metastasis properties may be partially by targeting MTHFR and that they exert anticancer effects in hepatocellular carcinoma cells.

The title should be changed accordingly, since rescue experiments were not performed; maybe "regulating" instead of "directly targeting"

 

Main points (numbers of points refer to the previous revision letter points):

 

2. Figure 2 legend: what do the letters used over the histogram bars indicate?

Because there are three bar graphs in Figure 2, one letter represents a bar graph, showing the proliferation status of a cell line. This can be clearly stated in the description to avoid confusion. Different letters indicate a significant difference in the histogram bars.

It is still not clear to which p value they correspond and how the comparison between the different treatments and time points has been performed.

 

3. Figure 3g: miR-149-5p was not used to determine MTHFR mRNA levels, do the authors have an explanation?

Because of the instability of RNA, the transcription level of MTHFR after transfection with miR-149-5p was not detected. But the protein level results were ideal and sufficiently supportive, so we did not have the results of RNA supplementation.

Why was it possible to detect MTHFR mRNA levels in the other two cell lines?

 

5. As stated in the discussion but without experimental data included in the manuscript (line 266), does MTHFR regulation affect PDCD4 and TP53INP1 in HCC cell lines?

Modified in lines 272-273.

A simple qPCR following siRNA-mediated MTHFR downregulation would have answered to this question.

 

6. Are PDCD4 and TP53INP1 involved in the regulation of proliferation and metastasis of HCC cells? The results included in this manuscript do not support this conclusion (line 268)

Modified in lines 238 and 272-273.

The authors should add any citations which describe the involvement of these two proteins in proliferation and metastasis in HCC to support their hypothesis.

 

7. The results do not demonstrate that miR-22 and miR-149 can inhibit cell proliferation and metastasis by downregulating MTHFR expression, no rescue experiments are included in the study.

Results 3.2. MiR-22-3p/miR-149-5p inhibit hepatocellular carcinoma cells proliferation, figure 1

Results 3.4. Transfection of miR-22-3p/miR-149-5p mimic suppresses migration and invasion in HCC cells in vitro, figure 4

Results 3.5. Transfection of miR-22-3p/miR-149-5p inhibitor promote migration in HCC cells in vitro, figure 5

Results 3.6. Silencing MTHFR inhibit migration in HCC cell lines, figure 5

Based on the above results, it can be confirmed that miR-22-3p and miR-149-5p inhibit the migration and that the invasion properties of HCC may be partially able to be achieved through downregulating MTHFR. Modified in lines 231-233.

The authors should change also the conclusions in the Discussion section (Line 273-276), they can only speculate that miR-22-3p and miR-149-5p inhibit the migration and  invasion of HCC cells by regulating MTHFR.

 

9. Line 282: there are no evidences that MTHFR affects TP53INP1 and PDCD4 in this study. Or has it been demonstrated in other studies?

We have marked in the text: Data not show here.

I cannot find the modified text

Author Response

Thanks to the reviewers for their comments, we have made corresponding revisions on the manuscript. including the following:

1. conclusion and title: They have all been revised based on comments.
2. Figure 2: It has been revised.
3. Figure 3g: Why was it possible to detect MTHFR mRNA levels in the other two cell lines?

Among the three cell lines in this experiment, only the transcription detection of HL-7702 transfected miR-149-5p was unsuccessful due to experimental operation problems. But the results at the protein level were ideal and sufficiently supportive, so we did not have results for RNA supplementation.

4. As stated in the discussion but without experimental data included in the manuscript (line 266), does MTHFR regulation affect PDCD4 and TP53INP1 in HCC cell lines? A simple qPCR following siRNA-mediated MTHFR downregulation would have answered to this question.

Due to the time limit of the manuscript revision, it was too late to make up the qPCR experiment. Thank you for your comments, we will further study this part in the future.

5. Are PDCD4 and TP53INP1 involved in the regulation of proliferation and metastasis of HCC cells? The results included in this manuscript do not support this conclusion (line 268). The authors should add any citations which describe the involvement of these two proteins in proliferation and metastasis in HCC to support their hypothesis.

Modified in lines 287-289.

6. The authors should change also the conclusions in the Discussion section (Line 273-276), they can only speculate that miR-22-3p and miR-149-5p inhibit the migration and  invasion of HCC cells by regulating MTHFR.

It has been revised in line 273-274.

7. Line 282: there are no evidences that MTHFR affects TP53INP1 and PDCD4 in this study. Or has it been demonstrated in other studies? I cannot find the modified text.

We have marked in line292: Data not show here.

Reviewer 2 Report

- Based on my previous criticism, the authors dampened the claim that the inactivation of MTHFR gene is required for tumor suppressor activity of miR-22-3p and miR-149-5p (since the experiment of functional rescue has not been performed) and demonstrated a role for MTHFR in cell migration. (Of note, in this experiment, the modulation of PDCD4 and TP53INP1 expression could have been assessed). However, the title is still incorrect. The expression “directly” should be removed (both in the title and in the abstract) since the direct regulation of MTHFR expression by miRNAs has not been demonstrated in this paper (but in a previously published work).

- Refs #8 and #9 have been still maintained although these papers do not report the role of MTHFR in HCC. Other references could be added

- Ref #10 supports the authors’ conclusions since the indicated paper shows a correlation between reduced MTHFR expression and low risk for late-stage HCC/increased survival of patients. However, the expression “Reduced MTHFR activities are associated with high-risk for the development of HCC” has not been explained and it is not supported by literature data.

- Letters “a”, “b” and “c” in figure 2 should be changed with the corresponding “p values”.

 

Author Response

Thank you for the comments, the title, abstract, references 8-10 and figure 2 have been revised.

Reviewer 3 Report

Some weaknesses remain in the revised version of the manuscript. While I appreciate the answers given by the authors to my different major comments, I would like these answers to be included in the manuscript. Indeed, I believe that these answers sheld light on important points associated with the way figure-panels are designed and experiments reported. This extra-information may ease the reading of the entire manuscript and help naive readers fully appreciate the work and the limitations.

Author Response

Thank you for the comments, the title, abstract, references and figures have been revised.

Round 3

Reviewer 1 Report

The authors replied to all my concerns.

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

In this paper the authors aimed at investigating the anticancer role of miR-22-3p and miR-149-5p. To this purpose, they overexpress miRNA mimics in hepatocellular carcinoma cells and normal hepatocytes demonstrating that ectopic miR-22-3p and miR-149-5p i) inhibit cell proliferation, migration and invasion of transformed cells, ii) downregulate Methylenetetrahydrofolate reductase (MTHFR) gene expression and upregulate TP53INP1 and PDCD4 gene expression. These effects can be observed in hepatocellular carcinoma cells but not in normal hepatocytes suggesting miR-22-3p and miR-149-5p as potential tools for the treatment of hepatocellular carcinoma. 

The manuscript presents several critical points.

Major concerns:

• The inhibition of tumor growth and metastasis by miR-22-3p and miR-149-5p (claimed by the authors in the title and in Discussion) has not been demonstrated since all experiments (cell proliferation/migration/invasion) have been carried out in cell lines and not in vivo. In particular, the invasion process is only one step of metastasis. The text should be therefore modified accordingly.
• The role of MTHFR in mediating the anticancer role of miRNAs has not been demonstrated (although it has been claimed in the title). The authors should have verified the impairment of anticancer effects of miRNA mimics upon silencing of MTHFR gene. In my opinion, this experiment is needed since data reported in this study are not new enough compared to those previously published  by the same authors (Li et al., 2017). In particular, i) the direct regulation of MTHFR gene expression by MiR-22-3p/miR-149-5p, ii) the correlation between miR-22-3p/miR-149-5p and MTHFR/Tp53INP1/PDCD4 expression and iii) the differential regulation of MTHFR expression in normal and cancerous human hepatocytes, have been previously demonstrated.
• The authors state that: “We also found that the inhibitor of miR-22-3p or miR-149-5p were respectively/both transfected in both cell lines, and the expression of MTHFR wasn’t restored in HCC cell line”. It can be assumed that HCC cells have high level of MTHFR and low levels of miR-22-3p or miR-149-5p. The experiment of miRNA inhibition should have been carried out in normal hepatocytes.
• The inhibition of cell proliferation by miRNA mimics in HCC cell lines is not impressive and limited to 72h post-transfection. Similarly, the decrease of MTHFR protein expression in WB of Figure 2C is slight. The same WB could be performed at 72h post-transfection and also in HepG2 cells. The number of independent experiments of transfection and Western Blot carried out and utilized for statistical analyses should be reported to allow a better evaluation of these results.
• The model in Figure 5 does not include only the data presented in this work. If the authors want to suggest additional pathways, they have to indicate more clearly the data that have been collected.
• The manuscript suffers from a low quality of the language (grammar errors, repetitions, incorrect sentences):

- Abstract: data should be more clearly described and repetitions should be avoided

- The following sentences should be revised:

Line 35: “Once the HCC progresses, the HCC progresses rapidly…”

Lines 85-86: “and their subsequent CCK-8 cell proliferation assay”

Lines 27 and 279: ectopic miR-22-3p/miR-149-5p could be used as a potential tool (not target) for the treatment of hepatocellular carcinoma

Lines 152-158

Lines 168-176

- Some sentences are unfinished or have repetitions (just to give an example: Lines 152-158)

- Grammar errors are present in the manuscript (e.g.: “controlled” in figure 1 legend instead of “control”; “decreased” in line 173 instead of “decreased”) as well as incorrect sentences (e.g. “Reduced MTHFR activities are associated with a high-risk for the development of HCC” in lines 62-63)

- The sentence “MTHFR expression by miR-22-3p and miR-149-5p might inhibit promoter methylation to positively regulate TP53INP1 and PDCD4, thus promoting HCC cell apoptosis” is not clear. No evidence of apoptosis has been reported in Results.

 

Minor points:

• Introduction (lines 38-42): the description of HCC treatments in brackets can be removed and replaced by references
• References about the role of miR-22 and mir-149 in cancer should be moved from Discussion to Introduction
• The quality of figures should be improved (e.g. control samples should precede experimental samples)
• The meaning of (a), (b) and (c) letters on the top of each column in figure 1 should be explained

Author Response

Dear reviewer：

 We carefully studied the opinions of the two reviewers and found that the article has many shortcomings. Recently, we have been adding experiments and modifying the language of the article in accordance with the opinions. Due to the experiment cycle problem, it could not be completed within the specified time, so I submitted an extension application to the editorial department. We also want to revise the article more perfect, hope to understand and give support, thank you!

Reviewer 2 Report

The paper by Li et al.

A deep revision of the entire manuscript by an English mothertongue is required, since a lot of grammar mistakes and confused sentences are present.

The abstract need to be reformulated since some sentences didn't adequately summarize the study. Moreover, the explanation of reported acronyms need to be written in the abstract.

In the section 2.2 the miRNAs sequences transfected in cells need to be specified.

Line 91 the sentence between brackets is quite unusual in the methods. Is it necessary to be specified?

In the section 2.4 Please add the miRNA Specific qPCR Primers used in the study.

It is not clear how the authors have controlled  the successful of miRNA transfection before doing the experiments. Maybe they could add some explanation in the methods section.

In fig. 1 the authors reported cell viability obtained by means of CCK8 assay. I think it could be better explain cell viability as % with respect to control rather than Abs units. Moreover, it is not clear if the authors used miRN-NC-mimic as control or not. I think that a control without transfection need to be added and also in the figures NC cells need to be put as first histogram and not as last since it is control. Did they also check cell viability and gene expression  in control non-transfected cell lines? 

The authors have to provide the entire Western blot images as support files.

Please check the text to be sure that all the acronyms along the manuscript have the right explanation.

Author Response

Dear reviewer：

 We carefully studied the opinions of the two reviewers and found that the article has many shortcomings. Recently, we have been adding experiments and modifying the language of the article in accordance with the opinions. Due to the experiment cycle problem, it could not be completed within the specified time, so I submitted an extension application to the editorial department. We also want to revise the article more perfect, hope to understand and give support, thank you!