Integrated bulk RNA and single-cell analysis with experimental validation reveal oxidative stress-related diagnostic biomarkers for osteoporosis

Cheng Zhong; Liping Zhong

doi:10.1371/journal.pone.0322326

Peer Review History

Original SubmissionNovember 19, 2024
19 Nov 2024 Author Response https://doi.org/10.1371/journal.pone.0322326.r001
20 Dec 2024 Decision Letter - Liangliang Xu, Editor PONE-D-24-52627Integrated Bulk RNA and Single-Cell Analysis with Experimental Validation Reveal Oxidative Stress-Related Diagnostic Biomarkers for OsteoporosisPLOS ONE Dear Dr. Zhong, 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 Feb 03 2025 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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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. 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 Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: The paper presents a new biomarker for early OP diagnosis and a therapeutic target.It is a topic of interest to the researchers in the related areas but the paper needs some improvement before acceptance for publication. My detailed comments are as follows: 1. The CHRM2/COL4A2 pathway was found in the paper works very well for the early OP diagnosis. On the other hand, A variety of early indicators for osteoporosis are currently available; where does CHRM2 have its advantages? Please detail this in the results or discussion section. 2. For the above reason, the presentation should be focused on the results. The transitions between each section are rather abrupt, and there are certain logical issues. The connection between the bioinformatics analysis and the subsequent experimental parts is poor; please make revisions. 3. In the cell experimentation section, the authors used siRNA to knock down COL4A2 in stem cells and observed their subsequent osteogenic differentiation ability. They also established a CHRM2 stem cell knockout model. Why not use this model to conduct experiments related to osteogenic differentiation? 4.A few sentences on the organization of the paper will be helpful in line 193-233. 5.The clarity of the figures in the article is poor; please replace them with clearer ones. 6.The English of your manuscript must be improved before resubmission. We strongly suggest that you obtain assistance from a colleague who is well-versed in English or whose native language is English. Reviewer #2: Comments to the Author This study focused on osteoporosis (OP). By integrating bulk RNA and single-cell analysis and combining with experimental verification, it explored the role of CHRM2 in osteogenic differentiation and its potential as a biomarker for OP. The research topic has significant clinical implications. Minor comments 1. In the Discussion section, although the research results were elaborated in relatively detailed manner, the explanations for some experimental results (such as the reasons for the expression changes of certain genes at different time points) were not in-depth and comprehensive enough. It is recommended to further combine relevant literature and research progress to conduct a more in-depth analysis and discussion on the experimental results. 2.Some sentences were not expressed clearly and smoothly enough, and there were some grammatical errors and improper use of professional terms. It is recommended to carefully proofread and modify them to improve the readability of the article. 3. The labels and descriptions of some charts and figures were not detailed enough. For example, some gene names and abbreviations were not explained in the charts and figures, which affected readers' understanding of the information in the charts and figures. It is recommended to optimize and improve the charts and figures. ****** 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: Yes: Lingli Ding ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
20 Feb 2025 Author Response Comment 1: The CHRM2/COL4A2 pathway was found in the paper works very well for the early OP diagnosis. On the other hand, A variety of early indicators for osteoporosis are currently available; where does CHRM2 have its advantages? Please detail this in the results or discussion section. Response: Thank you for this insightful question. While multiple early indicators for OP already exist—such as serum bone turnover markers (e.g., osteocalcin, CTX, and PINP), Wnt/β-catenin-related molecules (e.g., sclerostin), and various microRNAs—our data highlight several unique advantages of CHRM2: 1. Direct Link to Osteogenic Differentiation Unlike many markers that only reflect resorption or turnover, CHRM2 tightly aligns with the osteogenic process, as seen in its temporal expression changes during early to late osteoblast differentiation. 2. Integration With Immune and Oxidative Stress Pathways CHRM2’s regulatory role in both osteoblast maturation and immune cell interactions (via the CHRM2/COL4A2 pathway) may offer added diagnostic clarity, especially in early disease stages. 3. Potential Early Detection CHRM2’s dynamic expression pattern may detect subtle bone remodeling shifts before clinical OP manifestations, thus complementing more traditional markers. We have now added a concise comparison in the Discussion (page 19, lines 789–802) to underscore why CHRM2 may serve as a promising early indicator alongside other established pathways. Thank you for your valuable feedback. Comment 2. For the above reason, the presentation should be focused on the results. The transitions between each section are rather abrupt, and there are certain logical issues. The connection between the bioinformatics analysis and the subsequent experimental parts is poor; please make revisions. Response: Thank you for your comment regarding the abruptness of the section transitions and the poor connection between the bioinformatics analysis and the subsequent experimental parts. We have reorganized the Results to emphasize how the bioinformatics discoveries—particularly the identification of CHRM2/COL4A2—led us to conduct the follow-up experiments. Specifically, we have integrated clearer explanations of why the bioinformatic data pointed to CHRM2 as a candidate and how this insight shaped the in vitro validation. Additionally, we have used sequencing results to highlight our focus on the regulation of COL4A2 by CHRM2. By presenting the computational findings and experimental rationale in a more straightforward sequence, we believe the manuscript now flows more logically and underlines the significance of the CHRM2/COL4A2 pathway in early osteoporosis detection. We appreciate your feedback and hope these revisions address your concerns. . Comment 3. In the cell experimentation section, the authors used siRNA to knock down COL4A2 in stem cells and observed their subsequent osteogenic differentiation ability. They also established a CHRM2 stem cell knockout model. Why not use this model to conduct experiments related to osteogenic differentiation? Response: Thank you for raising this point. Initially, we focused on siRNA-mediated COL4A2 knockdown to delineate its specific role in osteogenic differentiation. After these experiments demonstrated the negative regulatory effect of COL4A2 on osteogenesis, we established the CHRM2 knockout model to explore the broader implications of the CHRM2/COL4A2 pathway in cell proliferation and osteogenic processes. In the manuscript, we performed Western blot, flow cytometry, ALP activity assay, and Alizarin Red staining experiments to compare the knockdown group with the control group. The experimental results confirmed that CHRM2 is a negative regulator in the osteogenic differentiation process. We appreciate this suggestion and will integrate these experiments into our future work. Figure 7. The effects of CHRM2 knockdown on osteogenic differentiation and cell proliferation. (A) The expression of CHRM2 was significantly reduced in cells knocked down with Si-CHRM2. (B) The RT-qPCR results showed that the expression of CHRM2 in cells with Si-CHRM2 knockdown decreased significantly. (C) The cell proliferation were measured by OD value at 450 nm, and the observation time points were at 0, 24, 48, 72 and 96 hours. (D) The protein expression levels of osteogenic markers RUNX2, OPN, and Osterix in Si-CHRM2 knockdown cells and control cells by western blot. (E) Flow cytometry of cell cycle in the control group and the experimental group. (F) ALP staining in the Si-CHRM2 experimental group. (G) Alizarin red staining. p < 0.05; p < 0.01; *p < 0.001; ns: no significance. Comment 4. A few sentences on the organization of the paper will be helpful in line 193-233. Response: Thank you for pointing this out. This new text provides a concise overview of the paper’s organization, showing how each section builds on the previous one. We believe this adjustment will help readers follow the logical progression from data analysis to experimental validation more smoothly. Comment 5. The clarity of the figures in the article is poor; please replace them with clearer ones. Response: Thank you for bringing the figure clarity to our attention. We have replaced the original figures with higher-resolution versions (at least 300 dpi) and adjusted the formatting to improve visibility and detail. We hope these revised figures will better convey our results and enhance the overall readability of our manuscript. Comment 6. The English of your manuscript must be improved before resubmission. We strongly suggest that you obtain assistance from a colleague who is well-versed in English or whose native language is English. Response: Thank you for your recommendation. We have thoroughly revised the manuscript with the assistance of a native English speaker to ensure accuracy, clarity, and fluency. We appreciate your feedback and believe that our revisions address the language concerns. If there are any additional improvements you would suggest, we would be glad to make further adjustments. Response to Reviewer#2: Comment 1: In the Discussion section, although the research results were elaborated in relatively detailed manner, the explanations for some experimental results (such as the reasons for the expression changes of certain genes at different time points) were not in-depth and comprehensive enough. It is recommended to further combine relevant literature and research progress to conduct a more in-depth analysis and discussion on the experimental results. Response: Thank you for your valuable comments regarding the depth and comprehensiveness of the Discussion section, especially concerning the temporal changes in gene expression. We have revised the Discussion to include additional literature and a more thorough analysis of the molecular mechanisms behind these observations. We appreciate the reviewer’s feedback and trust that these revisions and additional references will address the request for a more comprehensive and literature-supported discussion of our time-course findings. Comment 2: Some sentences were not expressed clearly and smoothly enough, and there were some grammatical errors and improper use of professional terms. It is recommended to carefully proofread and modify them to improve the readability of the article. Response: Thank you for highlighting the need for clearer language and more precise terminology. We have meticulously reviewed and revised the manuscript to address grammatical issues and refine the use of specialized terms, ensuring that our phrasing is both accurate and easily understood by readers. We appreciate your feedback and believe these improvements enhance the overall clarity and readability of our work. Comment 3: The labels and descriptions of some charts and figures were not detailed enough. For example, some gene names and abbreviations were not explained in the charts and figures, which affected readers' understanding of the information in the charts and figures. It is recommended to optimize and improve the charts and figures. Response: Thank you for pointing out the need for more detailed labeling and annotation in our charts and figures. We have revised the figure legends to include clear explanations of all gene names, abbreviations, and relevant symbols. Additionally, we have updated the figure labels to provide sufficient detail for readers to interpret the data accurately. We appreciate your feedback and believe these adjustments will improve the clarity and accessibility of our visual materials. Attachments Attachment Submitted filename: Response to Reviewers.docx* https://doi.org/10.1371/journal.pone.0322326.r003
19 Mar 2025 Decision Letter - Liangliang Xu, Editor <p>Integrated Bulk RNA and Single-Cell Analysis with Experimental Validation Reveal Oxidative Stress-Related Diagnostic Biomarkers for Osteoporosis PONE-D-24-52627R1 Dear Dr. Zhong, 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. If you have any questions relating to publication charges, 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, Liangliang Xu Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: (No Response) ****** 7. 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0322326.r004
Formally Accepted
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