Comprehensive Analysis of lncRNA and mRNA Reveals the Effect of ZBED6 on Spleen Growth in Pigs
Round 1
Reviewer 1 Report
The present manuscript describes the role of ZBED6 in the splenic growth of pig by regulating IGF2 (insulin- 16 like growth factor 2). Although lots of published literatures showed the role of ZBED6 in skeletal muscle growth, its role in splenic growth has not been investigated. Therefore, this is a novel approach to prove the proposed hypothesis. In the present investigation, only qRT-PCR has been done using GAPDH and β actin as reference gene controls. The use of suitable reference genes is needed for effective qPCR data analyses. Thus, the expression patterns of other common qPCR reference genes such as B2M, HMBS, HPRT1, ACTB, HSP90, HMBS, TBP, SDHA POLR2A, ACAC, and YWHAZ should be explored for their use as the appropriate reference gene control rather than using GAPDH and β actin (most frequently used). The use of invalidated reference gene in normalization might lead to unreliable conclusions while dealing with tissue samples. Kindly refer to the published article regarding the use of a set of reference housekeeping genes for the normalization of RT-qPCR data from the intestine of piglets (Wang et al., 2018) where they reported GAPDH and beta actin as the least stable genes. The tables and figures are adequate. However, more references of recent reports can be cited. Besides qRT-PCR analysis, the demonstration of ZBED6 in splenic tissue (IHC/IFT) will strengthen the findings. Overall, the quality of the manuscript is good after changes in methodology.
Author Response
Response: Thank you for your good suggestions.
- According to your suggestion, we added the RT-qPCRusing B2M and HMBS as reference gene control. The results and primer sequences (Supplementary Table 1) have been updated.
- b) Thanks. According to your suggestion, we cited some references of recent reports.
- Wang, J.; Chen, J.-F.; Ma, Q.; Mo, D.-L.; Sun, J.-J.; Ren, Q.-L.; Zhang, J.-Q.; Lu, Q.-X.; Xing, B.-S. Identification and Characterization of CircRNAs Related to Meat Quality during Embryonic Development of the Longissimus Dorsi Muscle in Two Pig Breeds. Front. Genet.2022, 13, 1019687, doi:10.3389/fgene.2022.1019687.
- Curtis, D. Weighted Burden Analysis in 200,000 Exome-Sequenced Subjects Characterises Rare Variant Effects on BMI. Int. J. Obes. (Lond).2022, 46, 782–792, doi:10.1038/s41366-021-01053-4.
- Zou, H.; Yu, D.; Yao, S.; Ding, F.; Li, J.; Li, L.; Li, X.; Zhao, S.; Pang, Y.; Hao, H.; et al. Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System. Genes (Basel).2022, 13, doi:10.3390/genes13071132.
- Zhu, S.; Zhang, J.; Jiang, X.; Wang, W.; Chen, Y.Q. Free Fatty Acid Receptor 4 Deletion Attenuates Colitis by Modulating Treg Cells via ZBED6-IL33 Pathway. eBioMedicine2022, 80, 104060, doi:10.1016/j.ebiom.2022.104060.
c). Thank you very much for your opinion. We are very sorry. Due to lake of anti-ZBED6 antibody now, the experiment of ZBED6 expression in splenic tissue (IHC/IFT) was not carried out. But in our previous study “Wang, D.; Pan, D.; Xie, B.; Wang, S.; Xing, X.; Liu, X.; Ma, Y.; Andersson, L.; Wu, J.; Jiang, L. Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets. PLoS Genet. 2021, 17, 1–18, doi:10.1371/journal.pgen.1009862.”, we had illuminated how the ZBED6 KO pigs were generated (Fig1A-1D), and ZBED6 expression in ZBED6 KO pigs (Fig1E-1F). The explanations are following: A CRISPR/Cas9-mediated, nonhomologous end-joining-independent integration strategy was developed to knock out ZBED6 in Bama miniature pigs (Fig1A-B). This 1-bp deletion (one-base pair deletion at position 1320-T) in ZBED6 KO pigs is a frameshift mutation(Fig1C), introducing the transcript was out of frame after codon 134 and a premature stop occurs at codon 224. This produces a short truncated protein (223 amino acids out of 981 amino acids in the full length protein = 22.7%) that did not maintain either of the two BED-DNA binding domains (Fig1D). qPCR analysis showed that this 1-bp deletion in ZBED6 KO pigs did not change ZBED6 transcripts (Fig1E). Western blotting showed undetectable ZBED6 protein in ZBED6 KO pigs (Fig1F). These results suggested that we successfully generated ZBED6 KO pigs.
In addition, as a transcription repressor of IGF2, ZBED6 knockout could up-regulated the expression of IGF2. So we also confirmed the up-regulated IGF2 expression in ZBED6 KO pigs in this study (Fig3F and Fig6).
And your suggestion has brought us a new idea for further study.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this article, the authors investigated the role of ZBED6 in spleen growth through comprehensive analysis of lncRNA and mRNA. The authors have done a good job and interesting to following.
There are some minor points/comments:
1. “Reveals” in the title “Comprehensive analysis of lncRNA and mRNA Reveals the effect of ZBED6 on spleen growth in pigs” should be “reveals”.
2. This article mainly investigated spleen growth, the authors should remove the part about immune response. For instance, functional enrichment analysis of DEGs was mainly enriched myofibril assembly, sarcomere, innate immune response, immune response and defense response.
3. Line 27, – "252 cis- and 109 trans-acting target genes were predicted." This sentence is misleading, whose target genes ?
4. Gene names should be described in italics.
5. Lines 68-70, –Spleen traits of WT and ZBED6 KO pigs (found and F4 generation) were measured to confirm the function of ZBED6 in regulating the spleen growth. Found should be founder.
6. Figure 3 KO or ZBED6 KO?
7. Lines 181-182 -The Q30 which can evaluate the quality of clean reads were above 86.865 in all the libraries. What unit of Q30 is ?
8. Line217, -The IGF2 level of spleen in ZBED6 KO pigs was 2.8-fold higher than that of WT pigs (WT: ZBED6 KO = 43.6: 124) (Fig 3F), suggested that ZBED6-IGF2 mainly played a role on spleen development in pigs. Suggested or suggesting?
9. The paragraph format of references were incorrect. Please align text.
10. The p in P values should be italic and lowercase.
11. Line 138, -GO enrichment analysis was carried out using the g:Profiler web server. Please include the link to the g:Profiler web server.
12. Uppercase and lowercase letters in the subtopics of results should be united.
For instance, “3.2. Whole Transcriptome Sequencing Results”
13. Figure 4 go pathway or go pathways? The initial of every pathway should be uppercase.
14. I am not clear about what the colors of Figure 5 D mean.
15. Please modify the Figure 6, and include the original data which likes Figures 1A-B.
Author Response
1.“Reveals” in the title “Comprehensive analysis of lncRNA and mRNA Reveals the effect of ZBED6 on spleen growth in pigs” should be “reveals”.
Response: Thank you for your good suggestions. We are very sorry for the mistake in the title. The title has been modified to “Comprehensive analysis of lncRNA and mRNA reveals the effect of ZBED6 on spleen growth in pigs ”.
- This articlemainly investigated spleen growth, the authors should remove the part about immune response. For instance, functional enrichment analysis of DEGs was mainly enriched myofibril assembly, sarcomere, innate immune response, immune response and defense response.
Response: Thanks. We has removed the part about immune response.
Lines 25-26, in abstract: the sentence “Functional enrichment analysis of DEGs was mainly enriched myofibril assembly, sarcomere, innate immune response, immune response and defense response.” has been modified to “Functional enrichment analysis of DEGs was mainly enriched myofibril assembly and sarcomere.”
Lines 227-229, in results, the sentence “Most of DEGs were enriched in the growth pathway and immune process, such as myofibril assembly, sarcomere, innate immune response, immune response and defense response (Fig 4).” has been modified to “Most of DEGs were enriched in the growth pathway, such as myofibril assembly and sarcomere (Fig 4).”
- Line 27, – "252 cis- and 109 trans-acting targetgenes were predicted." This sentence is misleading, whose target genes ?
Response: Thanks. It’s the target genes of DElncRNAs. The sentence “252 cis- and 109 trans-acting target genes were predicted.” has been modified to “252 cis- and 109 trans-acting target genes of 82 DElncRNAs were predicted. ”
- Gene names should be described in italics.
Response: Thanks. The gene names have been modified and described in italics.
- Lines 68-70, –Spleen traits of WT and ZBED6 KO pigs (found and F4 generation) were measured to confirm the function of ZBED6 in regulating the spleen growth.Found should be founder.
Response: Thanks. It should be founder.
The sentence “Spleen traits of WT and ZBED6 KO pigs (found and F4 generation) were measured to confirm the function of ZBED6 in regulating the spleen growth.” has been modified to “Spleen traits of WT and ZBED6 KO pigs (founder and F4 generation) were measured to confirm the function of ZBED6 in regulating the spleen growth.”
- Figure3 KO or ZBED6 KO?
Response: Thanks. It should be ZBED6 KO in Figure 3. We have modified KO to ZBED6 KO and a new version of Figure 3 has been provided.
- Lines 181-182 -The Q30 which can evaluate the quality of clean reads were above 86.865 in all the libraries. What unit of Q30 is ?
Response: Thanks. The unit of Q30 is %. We have added the unit of Q30.
“The Q30 which can evaluate the quality of clean reads were above 86.865 in all the libraries” has been modified to “The Q30 which can evaluate the quality of clean reads were above 86.865% in all the libraries”
- Line217, -The IGF2 level of spleen in ZBED6 KO pigs was 2.8-fold higher than that of WT pigs (WT: ZBED6 KO = 43.6: 124) (Fig 3F), suggested that ZBED6-IGF2 mainly played a role on spleen development in pigs.Suggested or suggesting?
Response: Thanks. It should be suggesting. “The IGF2 level of spleen in ZBED6 KO pigs was 2.8-fold higher than that of WT pigs (WT: ZBED6 KO = 43.6: 124) (Fig 3F), suggested that ZBED6-IGF2 mainly played a role on spleen development in pigs” has been modified to “The IGF2 level of spleen in ZBED6 KO pigs was 2.8-fold higher than that of WT pigs (WT: ZBED6 KO = 43.6: 124) (Fig 3F), suggesting that ZBED6-IGF2 mainly played a role on spleen development in pigs” .
- The paragraph format of references were incorrect.Please align text.
Response: Thanks. We have aligned the text of references.
- The p in P valuesshould be italic and lowercase.
Response: Thanks. We have modified the “p” in P values to “p”.
- Line 138, -GO enrichment analysis was carried out using the g:Profiler web server. Please include the link to the g:Profiler web server.
Response: Thanks. We have added the link to the g:Profiler web server.
“GO enrichment analysis was carried out using the g:Profiler web server” has been modified to “GO enrichment analysis was carried out using the g:Profiler web server (https://biit.cs.ut.ee/gprofiler/)” .
- Uppercase and lowercase letters in the subtopics of results should be united. For instance, “2. Whole Transcriptome Sequencing Results”
Response: Thanks. We have modified and ensure the subtopics of results united.
- Figure4 go pathway or go pathways? The initial of every pathway should be uppercase.
Response: Thanks. It should be “go pathways” and uppercase of initial of every pathway in Figure 4, and we have modified the mistakes in Figure 4. A new version of Figure 4 has been provided.
- I am not clear about what the colors of Figure 5 D mean.
Response: Thanks. The red means the key gene ACTN2, the green means known lncRNAs, and the gray means potentially_novel lncRNA. We have added this part to the legend of Figure 5D.
- Please modify the Figure 6, and include the original data which likes Figures 1A-B.
Response: Thanks. We have modified Figure 6 and include the original data.
Author Response File: Author Response.docx
