MiR-31 improves spinal cord injury in mice by promoting the migration of bone marrow mesenchymal stem cells

Yujuan Zhang; Lili Cao; Ruochen Du; Feng Tian; Xiao Li; Yitong Yuan; Chunfang Wang

doi:10.1371/journal.pone.0272499

Peer Review History

Original SubmissionDecember 24, 2021
25 Mar 2022 Decision Letter - Catherine Berry, Editor PONE-D-21-40503MiR-31 improves spinal cord injury in mice by promoting the migration of bone marrow mesenchymal stem cellsPLOS ONE 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. We have struggled to attain reviews for this article. The single review we have suggests major revision, as below. As editor, I have read the manuscript and also have suggestions on improvement. Should you decide to progress with all suggestions, we will look at a revised article. 1. The filed of miR-31 and MSCs is evidence for bone regeneration, however you do not refer to any of this research. Please widen your literature review of the activities of miR-31. 2. 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Revision 1
30 Apr 2022 Author Response 1、The filed of miR-31 and MSCs is evidence for bone regeneration, however you do not refer to any of this research. Please widen your literature review of the activities of miR-31. Thanks to the editors and reviewers for the questions I have answered as follows: MicroRNA-31 (miR-31), one of the many reported miRNAs, plays an important regulatory role in biological processes such as cell proliferation and migration. Studies have shown that miR-31, after activation, can act on RAS/MAPK, Hippo, JAK-STAT3, and other signaling pathways to regulate the proliferation and migration of the corresponding cells and thus promote the regeneration of skin tissue, intestinal epithelial tissue, skeletal muscle, and other damaged tissues. Current studies have been focusing on the effects of miR-31 on bone regeneration in mesenchymal stem cells. For example, in 2018 Jia et al. used gene chips to predict the regulation of mRNAs and miRNAs involved in osteogenic differentiation of ADSCs and found that miR-31 was highly correlated with osteogenesis in ADSCs. Similarly, Marupanthorn et al. found that ALP activity and osteogenic gene expression increased with increasing time during osteogenic differentiation in BMSCs treated with miR-31 inhibitors, a strategy that may be used to promote bone regeneration. The revised section is on lines 70-83 of the article. 2、Is the stability of miR-31 and the antagomiR verified in culture? Thanks to the editors and reviewers for the questions I have answered as follows: RNA interference (RNAi) is a natural cellular process that silences gene expression by promoting the degradation of mRNA. The RNAi mechanism was discovered in 1998 by Fire and Mello in caenorhabditis elegans, but this mechanism was not further developed until 2006. Currently, siRNAs have become an important tool for RNAi to achieve gene silencing and can be used to regulate the expression of target genes by transfecting them into cells. siRNA has also emerged as a potential and promising therapeutic platform and has been applied in many research areas. Conventional microRNA mimics or microRNA inhibitor is cumbersome to use and toxic to cells, which is not suitable for in vivo study and use in animals. Therefore, we attached a cholesterol moiety to the 3' end of miR-31 small nucleic acid molecule, which can enhance its ability to cross the cell membrane and enter the cell to play a regulatory role, and has higher stability in animal experiments and cellular experiments. Shu M et al. demonstrated that miR-335 transfected in glioma cells could be stably expressed in cells by PCR assay. Hu JZ et al. showed that the efficacy of miR-21 on SCI was still detectable 4 weeks after direct injection at SCI, indicating that it could be stably expressed in animals. Previous studies by our group have demonstrated that transfection of neural stem cells cultured in vitro with miR-31 verified its stable expression in neural stem cells by PCR and effectively restored motor function after SCI within 21 days after injection into mice with spinal cord injury. Due to the addition of a Cy3 light-emitting group, red fluorescence could be detected after transfection into the cells. Through this experiment, it was demonstrated that a large number of cells could emit red fluorescence after transfection with small nucleic acid molecules, and the results of fluorescence quantitative PCR showed that the expression of miR-31 was significantly upregulated in the miR-31agomir group (P�0.001) and downregulated in the miR-31antagomir group compared with the control group (P�0.01), indicating that the synthesized small nucleic acid molecules could effectively regulate the expression of miR-31 in BMSCs and thus determine the cell fate. And it was demonstrated that it could effectively restore the motor function of mice after SCI when injected with pretreated cells. The revised section is on lines 482-511 of the article. 3、All images/figures are of a low resolution, this must be improved. Thanks to the editors and reviewers for the questions I have answered as follows: All figures in the text have been revised and increased in resolution. The revised figure has been submitted separately. 4、the conclusion is too short and abrupt, this needs expansion and justification. Thanks to the editors and reviewers for the questions I have answered as follows: In this study, in vitro experiments demonstrated that at 48 h, 50�M miR-31agomir significantly promoted the proliferation of BMSCs, showing a time-dependent increase compared to 24 h, and its effect was verified by cell cycle analysis. The time-dependent promotion of miR-31 on the migration of BMSCs was demonstrated by migration assays. Thus, miR-31 was shown to promote the proliferation and migration of BMSCs. In the in vitro experiments, the relative fluorescence intensity of cells in mice demonstrated that the cells had the strongest migration ability at day 5 and 7, and the efficacy of miR-31 on the repair of motor function in SCI mice was demonstrated by BMS score.Therefore, this experiment demonstrated that miR-31 promoted the migration and proliferation of BMSCs by upregulating the expression of MMP-2 and CXCR4 to repair and improve the motor function of damaged tissues in SCI. However, whether it acts on CXCR4/AKT requires further studies to understand its detailed mechanism of action. The revised section is on lines 518-530 of the article. 5、Avoid keywords with the words used in the title, this method decreases a future search by your paper. Use keywords related to the theme. Thanks to the editors and reviewers for the questions I have answered as follows: Key words: Marrow mesenchymal stem cells (MSCs); Anti-inflammation; miR-31; Cell migration; Spinal cord injury (SCI) The revised section is on lines 41-42 of the article. 6、Let your text easy to the reader, for example: " (i) 1 mL syringe, (ii) aspirate the medium, (iii) collect the buffer, (iv) centrifuge and discard the supernatant, and (v) mix the precipitate". Thanks to the editors and reviewers for the questions I have answered as followsAnswer: Both ends of the bone were cut using tissue scissors, and following procedures were successively performed: (i) the bone marrow cavity was flushed three times using a 1 mL syringe; (ii) the culture medium containing bone marrow was collected; (iii) he supernatant was discard after centrifugation; (iv) the cell pellet was resuspended in DME/F-12 (Gibco, US); and (v) the resuspended cells were used to inoculate T25 culture flasks (Corning, US) to form a single cell layer. The revised section is on lines 112-117 of the article. 7、You applied parametric tests, however, your data are parametric? Shapiro-Wilk was tested ？ Thanks to the editors and reviewers for the questions I have answered as follows: We used the Student's T test to determine whether the data were normally distributed. Continuous data were expressed as mean ± standard deviation. One-way ANOVA and t-test were used for comparison between groups. All data were analyzed using SPSS 25.0 (IBM, NY, USA). Statistical graphs were produced by GraphPad Prism8 software (GraphPad，CA，USA), and a P < 0.05 was considered statistically significant. The revised section is on lines 231-236 of the article. 8、To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Thanks to the editors and reviewers for the questions I have answered as follows: During the experiments, 3% sodium pentobarbital was used for anesthesia to reduce animal pain, and mice were euthanized after high-dose anesthesia at the time of sampling. The revised section is on lines 101-103 of the article. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0272499.r002
21 Jul 2022 Decision Letter - Catherine Berry, Editor MiR-31 improves spinal cord injury in mice by promoting the migration of bone marrow mesenchymal stem cells PONE-D-21-40503R1 Dear Dr. Chunfang Wang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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. Kind regards, Catherine Berry, PhD, MSc, BSc Academic Editor PLOS ONE Additional Editor Comments (optional): The revised manuscript did take on board the suggestions given following the original submission by both the reviewer and the editor. All corrections were included and outlined clearly in the author's letter. https://doi.org/10.1371/journal.pone.0272499.r003
Formally Accepted
26 Aug 2022 Acceptance Letter - Catherine Berry, Editor PONE-D-21-40503R1 MiR-31 improves spinal cord injury in mice by promoting the migration of bone marrow mesenchymal stem cells Dear Dr. Wang: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Catherine Berry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0272499.r004

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