LNC297 promotes BMSCs differentiation and alleviates BHBA-induced inhibition through the miR-145/GAS7 axis

Tao Tang; Jing Zhou; Xianbo Jia; Jiahao Shao; Meigui Wang; Siqi Xia; Shuai Chen; Wenqiang Sun; Jie Wang; Song-jia Lai

doi:10.1371/journal.pone.0354035

Peer Review History

Original SubmissionApril 2, 2026
25 Jun 2026 Decision Letter - Gary Stein, Editor -->PONE-D-26-13971-->-->LNC297 Promotes BMSCs Differentiation and Alleviates BHBA-Induced Inhibition through the miR-145/GAS7 Axis-->-->PLOS One Dear Dr. Lai, 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. Please submit your revised manuscript by Aug 09 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes ****** -->5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: When ketosis occurs, dairy bovine muscle fibers can become damaged. It was unclear whether β‑hydroxybutyrate (BHBA), a type of ketone body, is involved in this phenomenon. This study examined the effects of BHBA on muscle fiber function and its molecular mechanisms in vitro. The results showed that treating bovine muscle satellite cells (BMSCs) with BHBA suppressed myogenic differentiation. Furthermore, the authors previously identified LNC297 and miR‑145 as having altered expression during instances of muscle fiber damage in ketosis bovines; similar changes were observed in this study. Subsequent database analyses and in vitro experiments involving miRNA mimics, siRNAs, and overexpression constructs demonstrated that: miR‑145 enhances the inhibitory effect of BHBA on myogenic differentiation; miR‑145 suppresses the expression of GAS7, a gene involved in muscle fiber growth and maintenance; LNC297 can negate the effects of BHBA by potentially binding to and inhibiting miR‑145. The data presented are clear, shedding light on aspects of BHBA’s effect and its underlying molecular mechanism. Beyond addressing issues associated with ketosis in dairy bovines, this study may also have implications for resolving human ketosis conditions in the future. However, there are a few issues in the manuscript that need to be addressed: 1.The first and second sentences in the abstract lack a clear logical connection; it is recommended to include relevant evidence to clarify the background. 2.Regarding GAS7 knockdown with siRNA: the title of Section 3.3 (“GAS7 alleviates the inhibitory effect of BHBA”) in Figure 3 does not appear to be supported by the data. It would be more convincing to test whether over-expression of GAS7 can counteract the effects of BHBA. The same suggestion applies to the experiments shown in Figure 4; using over‑expression instead of siRNA would better support the authors’ conclusions. 3."Replace the differentiation medium with an induction medium containing 4.8 mM BHBA and continue culturing for 12 hours." Why is a 12-hour treatment duration used? 4.Line 225 "ofmiR-145" should be changed to "of miR-145". 5.On line 244, "overexpressing LNC297" should be changed to "overexpressing LNC297", and on line 245, "LNC297promotes" should be changed to "LNC297 promotes". All relevant issues should be checked throughout the text. 6.The presence or absence of spaces between the reference citations and the main text needs to be uniformly standardized throughout the entire document. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. --> https://doi.org/10.1371/journal.pone.0354035.r001
Revision 1
1 Jul 2026 Author Response Reviewer 1: Dear Reviewer: On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript, we appreciate you and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “LNC297 Promotes BMSCs Differentiation and Alleviates BHBA-Induced Inhibition through the miR-145/GAS7 Axis” (PONE-D-26-13971). We have studied your and reviewer’s comments carefully and have made revision. The details are as follows: Comment #1: The first and second sentences in the abstract lack a clear logical connection; it is recommended to include relevant evidence to clarify the background. Response: Thank you for this helpful comment. We agree that the logical connection between the first two sentences was not sufficiently clear. To clarify the biological background, we have revised the opening of the Abstract by adding evidence that skeletal muscle is a major peripheral tissue involved in energy metabolism and is exposed to elevated circulating BHBA during ketosis. This revision better links ketosis-induced metabolic alterations with the potential role of BHBA in skeletal muscle regulation. The revised content is as follows: Comment #2: Regarding GAS7 knockdown with siRNA: the title of Section 3.3 (“GAS7 alleviates the inhibitory effect of BHBA”) in Figure 3 does not appear to be supported by the data. It would be more convincing to test whether over-expression of GAS7 can counteract the effects of BHBA. The same suggestion applies to the experiments shown in Figure 4; using over‑expression instead of siRNA would better support the authors’ conclusions. Response�We would like to thank the reviewer for their valuable comments on the experimental design. We understand the reviewer's concern regarding the title 'GAS7 alleviates the inhibitory effect of BHBA' and appreciate the suggestion to use GAS7 overexpression experiments to better validate our conclusions. The primary reason we chose to use siRNA to knock down GAS7 was to explore the effect of GAS7loss of function on BMSCs differentiation. The results show that when the expression of GAS7 in BMSCs is reduced, the expression of differentiation marker genes MYH7, MYOD1, and MYOG is downregulated, suggesting that GAS7 promotes BMSC differentiation. Some of the results in Section 3.2 demonstrate that BHBA inhibits BMSC differentiation, and when Battement is added while lowering GAS7 expression, this inhibition is further exacerbated. Therefore, we hypothesize that GAS7 alleviates the inhibitory effect of BHBA. Of course, these data may be better explained through a rescue experiment using overexpression. We are grateful for the reviewer’feedback on the experimental design flaws in this study. Finally, we would like to express our sincere gratitude to the reviewer for the valuable suggestions, which will provide precious insights for the design. Comment #3:"Replace the differentiation medium with an induction medium containing 4.8 mM BHBA and continue culturing for 12 hours." Why is a 12-hour treatment duration used? Response: We sincerely thank the reviewer for the valuable comments. Previous studies have shown that treatment of C2C12 cells with sodium β-hydroxybutyrate for 24 hours can promote myoblast proliferation and differentiation through energy metabolism and the GPR109A-mediated Ca²⁺-NFAT signaling pathway. However, in our study, we used β-hydroxybutyric acid (BHBA) and BMSCs, which differ from both the compound and the cell type used in prior work. Therefore, we shortened the treatment duration accordingly. In our experiments, a 12-hour treatment was sufficient to observe initial cellular responses and changes in the expression of differentiation markers. Although this duration may not fully support complete satellite cell proliferation and differentiation, it allows a subset of cells to enter the differentiation process and provides meaningful data regarding molecular marker dynamics. In our experimental design, the 12-hour treatment was intended as a preliminary induction period to assess early cellular responses within a short timeframe, and to inform future experiments involving longer treatment durations. We will clarify this point further in the Methods section, emphasizing that the 12-hour treatment time is a rational choice based on previous literature and preliminary results. Finally, we would like to express our gratitude to the reviewers for their valuable comments. The limitations of this research are also discussed in the article. Additionally, the methodology section (2.9) of the article also cites relevant literature to support the reasons for using this processing time. Comment #4: Line 225 "ofmiR-145" should be changed to "of miR-145". Response: Thank you very much for the reviewers' comments. The revisions have been made in the manuscript. Comment #5: On line 244, "overexpressing LNC297" should be changed to "overexpressing LNC297", and on line 245, "LNC297promotes" should be changed to "LNC297 promotes". All relevant issues should be checked throughout the text. Response: Thank you very much for the reviewers' comments. The revisions have been made in the manuscript. Comment #6: The presence or absence of spaces between the reference citations and the main text needs to be uniformly standardized throughout the entire document. Response: Thank you very much for the reviewers' comments. I have made the necessary revisions as per the journal's requirements. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0354035.r002
2 Jul 2026 Decision Letter - Gary Stein, Editor LNC297 Promotes BMSCs Differentiation and Alleviates BHBA-Induced Inhibition through the miR-145/GAS7 Axis PONE-D-26-13971R1 Dear Dr. Lai, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support. 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, Gary S. Stein Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0354035.r003
Formally Accepted
Acceptance Letter - Gary Stein, Editor PONE-D-26-13971R1 PLOS One Dear Dr. Lai, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@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. Gary S. Stein Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0354035.r004

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