Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells

Xiangqin Ji; Penglei Jiang; Juntao Luo; Mengjia Li; Yajing Bai; Junfang Zhang; Bingshe Han

doi:10.1371/journal.pone.0226905

Peer Review History

Original SubmissionJuly 24, 2019
19 Aug 2019 Decision Letter - Zongbin Cui, Editor PONE-D-19-20835 Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells PLOS ONE Dear Dr.Han, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Reviewer #1: The authors characterized the miRNA profiles in ZF4 cells acclimated to mild cold stress for 30 days and the time-matched controls maintained at 28 oC. They identified cold acclimation-associated miRNAs and provided evidence that some of these differentially expressed miRNAs were involved in cold resistance. The bioinformatic analysis pipeline is reasonable and the data is well organized. The following concerns should be addressed before publication. 1) In the abstract, it should be made clear that the list of putative target genes for the DE-miRNAs was used for GO and KEGG enrichment analyses. 2) Why did the authors select foldchange > 2.5 as the threshold for DE-miRNA? 3) Line 144, the title of Table 1 should be “Primer sequences ……” 4) In the miRNA transfection and cell viability assays, what are the sequence and quantity of miRNA mimics and inhibitors used for transfection? Only concentration of these stuffs was provided. How about the transfection efficiency? The authors analyzed relative expression of these molecules after transfection. However, the efficiency of transfection, namely the proportion of cells that express the exogenous molecules after transfection is very important for understanding the data of function investigation. 5) Line 164, how the reads without sRNA sequences, ranging from 18 to 35 nt in length, were identified? 6) The level of representative targets for the critical miRNAs should be analyzed to further support the conclusion that they are involved in cold acclimation by regulating the abundance or translation of their cognate targets. Reviewer #2: In this manuscript, the authors used RNA-seq to identify the miRNA roles in cold acclimation of ZF4 cell. Their previous study showed that DNA methylation and long non-coding RNAs (lncRNAs) are involved cold acclimation of zebrafish ZF4 cells when experimentally acclimated at 18˚C for 30 days, indicating that the authors have investigated the gene expression variation by RNA-seq methods using the ZF4 cell samples under two different temperatures. In this study, RNA-seq data analysis has revealed differential expression of genes including lncRNAs, microRNAs during cold acclimated at 18℃ for 30 days. Some concerns need to be addressed: 1) For the cold treatment, the ZF4 cell was seeded at 50% confluence and placed in an incubator at 18℃ and 5% CO2 for 30 days. Why di d you choose 50% confluence and how about the survival rates of cells after treatment for 30 days under low temperature? 2)To verify the miRNA function under cold pressure in ZF4 cells ,why did you choose to treat cells at 10℃,but not at 18℃，since the differential expression of miRNA was identified in samples at 18℃ and 28℃？Moreove, the measurement of cell viability with Trypan Blue staining under microscope need be provided and the MTT experiments for the cell viability may be verified with Trypan Blue staining. 3)To comclude that the miRNAs are involved in fish cold acclimation, the microRNA functions should be tested in zebrafish embryos or larvae to answer if these microRNAs affect the early development of zebrafish or play a role in cold acclimation. ============================== We would appreciate receiving your revised manuscript by Oct 03 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Zongbin Cui, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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Revision 1
2 Oct 2019 Author Response List of Responses Dear Editors and Reviewers: Thanks for your comments concerning our manuscript entitled "Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells" (Manuscript Number: PONE-D-19-20835). Those comments are very helpful for improving our data and we have made revision. The major corrections and the responds to the reviewer are as following: Reviewer #1: The authors characterized the miRNA profiles in ZF4 cells acclimated to mild cold stress for 30 days and the time-matched controls maintained at 28℃. They identified cold acclimation-associated miRNAs and provided evidence that some of these differentially expressed miRNAs were involved in cold resistance. The bioinformatic analysis pipeline is reasonable and the data is well organized. The following concerns should be addressed before publication. Thank you for your time reviewing this manuscript. We have made an extensive modification in the revised manuscript. The changes to our manuscript are highlighted by using red colored text. We would be glad to respond to any further questions and comments. 1) In the abstract, it should be made clear that the list of putative target genes for the DE-miRNAs was used for GO and KEGG enrichment analyses. Reply: Thanks for your suggestion, the proper information has been provided in the revised abstract. 2) Why did the authors select foldchange > 2.5 as the threshold for DE-miRNA? Reply: It is true that many researchers select foldchange > 1.5 (1) , 2 (2) as the threshold for DE-miRNA. Here we screened 55 known DE-miRNAs with foldchange > 2, when 28 known DE-miRNAs obtained with foldchange > 2.5. So here we set a stricter standard to screen out the most relevant miRNAs involved in cold acclimatiom. In the revised version, we provide the information of all DE-miRNA with foldchange > 1.5 for the readers in the Supplementary Table S5. 3) Line 144, the title of Table 1 should be “Primer sequences ……” Reply: Thank you very much for pointing out this error. We have corrected this issue through the whole revised manuscript. 4) In the miRNA transfection and cell viability assays, what are the sequence and quantity of miRNA mimics and inhibitors used for transfection? Only concentration of these stuffs was provided. How about the transfection efficiency? The authors analyzed relative expression of these molecules after transfection. However, the efficiency of transfection, namely the proportion of cells that express the exogenous molecules after transfection is very important for understanding the data of function investigation. Reply: The sequences and quantity of miRNA mimics and inhibitors have been provided in the revised manuscript. The sequences of mimics and inhibitors are summarized in Supplementary Table S1. In this study, the transfection efficiency ranges from 75% to 85% based on our experiments using a fluorescence-labeled control miRNA. 5) Line 164, how the reads without sRNA sequences, ranging from 18 to 35 nt in length, were identified? Reply: Reads without sRNA sequences, ranging from 18 to 35 nt in length, were filtered using cutadapt. We have provided the information in the revised manuscript. 6) The level of representative targets for the critical miRNAs should be analyzed to further support the conclusion that they are involved in cold acclimation by regulating the abundance or translation of their cognate targets. Reply: Thanks for your suggestion. In the revised manuscript, we showed that after treatment of ZF4 cells with mimics/inhibitor of miR-100-3p or miR-16b. An inverse correlation between the expression profiles of miRNAs (dre-miR-100-3p, dre-miR-16b) and their target genes (atad5a, cyp2ae1, lamp1; rilp, atxn7, tnika, btbd9) was confirmed by RT-qPCR 24h after transfection. And this relationship is consistent with our previous RNA-Seq data. We have provided our new observations in Fig. 5 to support this point. Reviewer #2: In this manuscript, the authors used RNA-seq to identify the miRNA roles in cold acclimation of ZF4 cell. Their previous study showed that DNA methylation and long non-coding RNAs (lncRNAs) are involved cold acclimation of zebrafish ZF4 cells when experimentally acclimated at 18˚C for 30 days, indicating that the authors have investigated the gene expression variation by RNA-seq methods using the ZF4 cell samples under two different temperatures. In this study, RNA-seq data analysis has revealed differential expression of genes including lncRNAs, microRNAs during cold acclimated at 18℃ for 30 days. Some concerns need to be addressed: Thank you for your time reviewing this manuscript. We have made an extensive modification in the revised manuscript. The changes to our manuscript are highlighted by using red colored text. We would be glad to respond to any further questions and comments. 1) For the cold treatment, the ZF4 cell was seeded at 50% confluence and placed in an incubator at 18℃ and 5% CO2 for 30 days. Why did you choose 50% confluence and how about the survival rates of cells after treatment for 30 days under low temperature? Reply: For cold acclimation of ZF4 cells, acute exposure to 18℃ of ZF4 cells with confluence less than 50% showed significant growth inhibition and cell death. So we chose staring cold treatment at 50% confluence to make sure that most cells survive the whole process. Growth inhibition and slight cell death could be observed only in the first two weeks. After 30 days of treatment, the survival rates of cells are close to ZF4 cells cultured at 28℃. Detailed information can be found S1 Fig in reference (3). 2)To verify the miRNA function under cold pressure in ZF4 cells ,why did you choose to treat cells at 10℃,but not at 18℃，since the differential expression of miRNA was identified in samples at 18℃ and 28℃？Moreove, the measurement of cell viability with Trypan Blue staining under microscope need be provided and the MTT experiments for the cell viability may be verified with Trypan Blue staining. Reply: Thanks for your suggestion. In our study, ZF4 cells were cold acclimated at 18℃, then DE-miRNA were identified. Cold acclimated cells differ form control cells by showing enhanced cold tolerance under severe cold exposure (10℃ in this work). And ZF4 cells at 50% confluence only showed slight cell death at 18℃, so it is hard to see the role of DE-miRNA at 18℃. So we chose to treat cells at 10℃ to verify the miRNA function during cold acclimation in ZF4 cells. We have provided the microscope photos of Trypan Blue staining in Supplementary Fig.3 according to your suggestion. For our MTT assay kit, the attached instruction does not suggest its application at 10℃, and we observed that its application at RT/25℃ was likely disturbed by the cold-rewarming process, so we hesitated to provide this information for now. 3) To comclude that the miRNAs are involved in fish cold acclimation, the microRNA functions should be tested in zebrafish embryos or larvae to answer if these microRNAs affect the early development of zebrafish or play a role in cold acclimation. Reply: According your comments, we investigated the effect of one selected miRNA on the early development of zebrafish. Our data showed that microinjection of zebrafish zygotes with miR-100-3p mimics led to increased death from 6 hpf, while miR-100-3p inhibitor showed only slight effect on the survival and development of zebrafish zygotes. The data have been provided in Fig.6. We will work on more miRNA including miR-16b in our further research. Yours sincerely, Bingshe Han Bingshe Han, PhD Building A Room A117 College of Fisheries and Life Science, Shanghai Ocean University No.999 Hucheng Ring Road Shanghai 201306, China Tel:86-21-61900476/Fax:86-21-61900492 Email: bs-han @shou.edu.cn Reference: 1. Tang Z, Zhang L, Xu C, Yuan S, Zhang F, Zheng Y, et al. Uncovering small RNA-mediated responses to cold stress in a wheat thermosensitive genic male-sterile line by deep sequencing. Plant physiology. 2012;159(2):721-38. 2. Liu W, Cheng C, Chen F, Ni S, Lin Y, Lai Z. High-throughput sequencing of small RNAs revealed the diversified cold-responsive pathways during cold stress in the wild banana (Musa itinerans). BMC Plant Biol. 2018;18(1):308. 3. Han B, Li W, Chen Z, Xu Q, Luo J, Shi Y, et al. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure. PloS one. 2016;11(8):e0160358. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0226905.r002
28 Nov 2019 Decision Letter - Zongbin Cui, Editor PONE-D-19-20835R1 Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells PLOS ONE Dear Dr.Han, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Reviewer 2: The authors didn't show any evidence of the role of microRNAs in fish larvae, so they shoud change the conclusion that miRNAs are closely involved in cold acclimation in fish in the abstract. Does the miR-16b inhibitor have effects on fish cold acclimation? ============================== We would appreciate receiving your revised manuscript by Jan 12 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Zongbin Cui, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226905.r003
Revision 2
5 Dec 2019 Author Response List of Responses Dear Editors and Reviewers: Thanks for your comments concerning our manuscript entitled "Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells" (Manuscript Number: PONE-D-19-20835R1). These comments are very helpful for improving our data and we have made revision. We would be glad to respond to any further questions and comments. The major corrections and the responds to the reviewer are as following: Reviewer 2: The authors didn't show any evidence of the role of microRNAs in fish larvae, so they should change the conclusion that miRNAs are closely involved in cold acclimation in fish in the abstract. Does the miR-16b inhibitor have effects on fish cold acclimation? Reply: They should change the conclusion that miRNAs are closely involved in cold acclimation in fish in the abstract Thank you very much for pointing out this issue. We have corrected this issue in the abstract. Does the miR-16b inhibitor have effects on fish cold acclimation? It is a very good question. As you pointed out in your comment, for now we do not have the data about the effect of miR-16b inhibitor on fish larvae, so we can not show the effects of miR-16b inhibitor on fish cold acclimation directly. Our ongoing work shows that miR-16b inhibitor microinjection led to increased abnormal development of zebrafish, so those fish larvae are not appropriate for evaluation of the effect of miR-16b inhibitor on fish cold acclimation, because it is hard to attribute observed changes to miR-16b inhibitor or early development defects. Further study of the role of miR-16b in fish may need conditional knockdown of miRNA in zebrafish. The present data suggested the role of miR-16b in cold acclimation of ZH4 cells based on that miR-16b mimics enhanced cell survival under cold stress when miR-16b inhibitor led to decreased cell viability (Fig. 6B). So we hypothesized that miR-16b inhibitor may have the similar effect on fish cold acclimation. If you have any suggestion, we will be very glad to hear from you. Yours sincerely, Bingshe Han Bingshe Han, PhD Building A Room A117 College of Fisheries and Life Science, Shanghai Ocean University No.999 Hucheng Ring Road Shanghai 201306, China Tel:86-21-61900476/Fax:86-21-61900492 Email: bs-han @shou.edu.cn Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0226905.r004
10 Dec 2019 Decision Letter - Zongbin Cui, Editor Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells PONE-D-19-20835R2 Dear Dr. Han, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Zongbin Cui, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226905.r005
Formally Accepted
27 Dec 2019 Acceptance Letter - Zongbin Cui, Editor PONE-D-19-20835R2 Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells Dear Dr. Han: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Zongbin Cui Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226905.r006

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