Knockdown of miR-21-5P targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates in sulin resistance in polycystic ovary syndrom

Hengzhen He; Peng Ning; Xiaohong Chen; Jing Lin

doi:10.1371/journal.pone.0343735

Peer Review History

Original SubmissionOctober 7, 2025
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Thank you for stating in your Funding Statement: -->-->This work was supported by the Natural Science Foundation of Guangxi Province (grant number 2023GXNSFBA026257 and 2024GXNSFAA010146),  National Natural Science Foundation of China (grant number 82560972),the Guangxi Key R&D Program (grant number Guike AB19110022).Author contributions -->-->He Hengzhen conceived, designed, and conducted the experimental study; she also participated in data collection, project assembly, data analysis and interpretation, and manuscript writing. Ning Peng conducted the experimental study, data assembly, and analysis.Chen Xiaohong,Lin Jing conceived, designed, and conducted the experimental study and revised the manuscript.  All authors read and approved the final manuscript. -->--> -->-->Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. -->-->Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.-->--> -->-->3. 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The main authors' conclusions are that the studies miR regulates PDCD4 and secondary participates to insulin resistance of PCOS. The manuscript needs a deep proof-reading as contradictions appear. The style is somehow heterogeneous in the description of the figures. Following are some comments that need to be answered. Among the comments listed below, description of the results and discussion are sometimes mixed. For a better reading, this should be corrected. * Introduction: more citations are needed, mainly in the second paragraph. Last sentence of the introduction is too long and should be rephrased. Besides "Mechanism" appears alone... pleased correct that. * Material and methods section: - Establishment of the PCOS...: many repetitions are present. Please correct this point. - Grouping and lentivirus injection: 6 groups are indicated; however, are there 3 conditions (PCOS, shNC and sh-miRNA-21-5p) and 2 duplicates per condition ? In the abstract only 4 groups are mentioned. This needs to be clarified. - AlphaFold3...: Please use a more "scientific language" to described the process and not a step-by-step story telling. - primary granulocyte collection: i) a granulocyte is not the same cell type as a granulosa cell. Please correct as well the title of the paragraph; ii) the first sentence should be rephrased as it has no sense: rats were anesthetized before ovary collection; iii) the most important point is the following: how to be sure that only cells from granulosa have been isolated for avoiding contamination by various cells? How long the cells can be conserved without loosing their characteristics? - PDCD4 inhibitor treatment...: please indicate which ovarian granulosa cell line was used for the ex vivo experiments. * Results: - miRNA-21-5p Targets and Regulates PDCD4: This paragraph is intended to show a key point to confirm the hypothesis that miRNA regulates PDCD4. However, the mechanism is not clear: Fig 4D shows that the more miRNA-21-5P, the highest PDCD4 expression. Fig 4E shows a potential miRNA-21-5P interaction with the PDCD4. How the interaction of a miRNA with the protein may regulate the expression of the PDCD4 gene ?? - miRNA-21-5p Regulates Ovarian Granulosa Cell Proliferation and Apoptosis by Targeting PDCD4: it is stated that "a positive correlation between miRNA-21-5p expression and PDCD4 activation” is confirmed and “PDCD4 expression was significantly reduced in the si-miRNA-21-5p group”. However, this seems in contradiction with the first sentence of the introduction lettre indicating “knockdown of miR-21-5P up-regulates PDCD4 expression". Which part is correct? - Figures 1F and 4C are identical. This should be explained. If this is the case, one should be omitted. - miRNA-21-5p regulates ovarian granulosa cell proliferation... Line 3, it is indicated that there is a positive correlation between "miRNA-21-5p expression and PDCD4 activation". Actually PDCD4 accumulation has been shown, not activation. Please correct or explain. Minor points: - miR-21-5P or miRNA-21-5p? Please homogenize throughout the manuscript. - FSH and E2 results are in figure 2 not Figures 3. Please correct that point. - Legend figure 2: "silencing of serum LH secretion" is incorrect please correct to "decreasing" Figure 5A: in the Y-axis, it is indicated "relative PDCD4 expression". Relative to what? Please precise that point. There same comments coud be done for other figures. [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. 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.0343735.r001
Revision 1
1 Dec 2025 Author Response Dear Dr. Lobaccaro, We would like to express our sincere gratitude for your thoughtful and constructive comments on our manuscript entitled “Knockdown of miRNA-21-5p targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates insulin resistance in polycystic ovary syndrome” We apologize for the delay in submitting the revised version and greatly appreciate the opportunity to further improve our work. We have carefully considered each comment provided by the editors and reviewers, and the manuscript has been comprehensively revised accordingly. All modifications are clearly marked in red in the revised manuscript. Our detailed point-by-point responses are outlined below. 1. General Revisions 1.1 Language refinement and stylistic improvement The entire manuscript has undergone thorough language refinement to enhance clarity, coherence, and academic tone. In addition, the revised manuscript has been professionally edited by a native English speaker to ensure compliance with the journal’s linguistic standards. 1.2 Terminology standardization We appreciate the editor’s guidance. All occurrences of “miR-21-5p” have been uniformly updated to “miRNA-21-5p” to maintain consistency throughout the manuscript. 2. Introduction 2.1 Updated references and refinement of the concluding sentence Thank you for this helpful suggestion. We have incorporated additional references into the first and second paragraphs to strengthen the scientific context. The concluding sentence has been rephrased to enhance precision and readability. 3. Materials and Methods 3.1 PCOS model establishment and group clarification We appreciate the editor’s insightful comment. Redundant descriptions regarding the PCOS model have been removed to improve clarity. The earlier inconsistency in the abstract, which mentioned four groups, has been corrected. The revised manuscript now clearly indicates that five experimental groups were included, with detailed information provided in the Methods section. 3.2 Standardization of AlphaFold3 description The AlphaFold3 subsection has been fully rewritten to adopt a concise and scientifically appropriate style. Narrative, step-like descriptions have been removed to improve alignment with standard methodological reporting. 3.3 Correction and clarification of granulosa cell isolation We sincerely apologize for the previous misuse of terminology, and we thank the editor for pointing this out. “Granulocyte” has been corrected to “granulosa cells.” The isolation procedure has been described in greater detail, including steps taken to ensure cell purity and to preserve cellular characteristics during storage. 3.4 Clarification regarding PDCD4 inhibitor treatment We have added clear information regarding the ovarian granulosa cell line used in exogenous treatment experiments, thereby improving transparency and reproducibility of the experimental workflow. 4. Results 4.1 Regulatory relationship between miRNA-21-5p and PDCD4 We appreciate the editor’s insightful suggestion. This section has been rewritten to more clearly elucidate the targeting and regulatory mechanism of miRNA-21-5p on PDCD4. Moreover, co-immunoprecipitation (co-IP) data have been included to further substantiate this interaction. 4.2 Correction of previously inaccurate description We sincerely regret the earlier inaccuracies regarding the regulatory relationship between miRNA-21-5p and PDCD4. The inconsistency between the Introduction and Results has now been fully resolved. The Introduction correctly states that “knockdown of miRNA-21-5p leads to downregulation of PDCD4,” and the Results section provides a clearer explanation of this relationship. 4.3 Clarification regarding duplicated figures Due to an oversight, Figures 1F and 4C were inadvertently duplicated. We thank the editor for bringing this to our attention. One of the figures has been removed, and the corresponding text has been updated accordingly. 4.4 Revision of terminology describing PDCD4 expression The term “activation” has been refined to “increased expression,” which more accurately reflects the results presented in the figures. 5. Minor Revisions 5.1 Correction of figure references for FSH and E2 We appreciate the editor’s observation. The references to FSH and E2 in the text have been adjusted to correctly correspond to Figure 2. 5.2 Revision of the legend for Figure 2 As suggested, the legend for Figure 2 has been carefully revised for accuracy and clarity. 5.3 Clarification of the y-axis label in Figure 5A We thank the editor for identifying this issue. The y-axis label in Figure 5A now clearly states that “relative PDCD4 mRNA expression” is presented relative to the control group. 6. Additional Requirements 6.1 Updated funding statement All funding sources have been fully listed. The revised statement is as follows: “This study was supported by the Guangxi Natural Science Foundation (Nos. 2023GXNSFBA026257 and 2024GXNSFAA010146), the National Natural Science Foundation of China (No. 82560972), and the Guangxi Key Research and Development Program (Guike AB19110022). No additional external funding was received.” 6.2 Updated data availability statement The Data Availability Statement has been updated to indicate that all data will be made publicly available upon acceptance, in full compliance with the PLOS ONE data-sharing policy. The corresponding link has been added to the end of the manuscript. 6.3 ORCID iD verification The ORCID iD of the corresponding author has been successfully verified through the Editorial Manager system. 6.4 Ethical statement adjustment The ethical statement has been relocated to the Methods section and revised to conform to the journal’s guidelines. 6.5 Supporting Information updates Legends have been added to all Supporting Information files, and their citations in the main text have been corrected for consistency. Conclusion We express our sincere appreciation to the editor and reviewers for their detailed assessment and invaluable suggestions. As our research team must complete the final documentation for a funded project by the end of this month, we would be deeply grateful if the review and editorial decision could be expedited where feasible. We greatly appreciate your understanding and continued support. Sincerely, The Authors Attachments Attachment Submitted filename: Reply_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0343735.r002
5 Jan 2026 Decision Letter - Jean-Marc Lobaccaro, Editor Dear Dr. chen, 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 19 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. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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We look forward to receiving your revised manuscript. Kind regards, Jean-Marc A Lobaccaro, PhD Academic Editor PLOS One Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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 Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> 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 Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The authors have made most of the corrections necessary for the manuscript to be accepted, but this is not yet completely achieved. Some sentences still need to be corrected, either to improve readability or to avoid misunderstandings. There is also missing information. These minor corrections should be addressed before final acceptance. 1. The first paragraph of the Introduction still contains repetitions and syntactic heaviness that could be improved. Here is what could be suggested instead of the current third and fourth sentences of this first paragraph: “PCOS is considered the most likely cause of anovulatory infertility, where disturbances in sex hormone secretion, insulin resistance, and abnormal follicular development play a key role in its pathogenesis [4]. Insulin resistance is both a major pathological and clinical feature of polycystic ovary syndrome [5–7], making the development of an animal model highly significant for advancing research into this condition [8].” 2. In the Materials and Methods paragraph “Primary Granulosa Cell Collection,” there are misorganized arguments that could lead to misunderstanding: “Granulosa cells were extracted from rat ovaries and anesthetized by intraperitoneal injection of sodium pentobarbital (3% Pelltobarbitalum Natricum, Merck, Germany, 0.03 ml/100 g) according to a previous study.” Animals were anesthetized, not granulosa cells. 3. In the “RNA Immunoprecipitation (RIP) Assay” paragraph, the references for the antibodies used in the experiment are missing. 4. In the “Protein Extraction and Western Blot Analysis” paragraph, the title suggests an expression analysis of PDCD4; however, the text does not provide any corresponding information (e.g., the antibody used and its reference). 5. The name of the PDCD4 inhibitor, as well as the name of the company that supplied it, should be provided in the text the first time it is cited (Materials & Methods section). 6. Reference 9 is not appropriate. It does not deal with Western medicine and/or therapies for treating PCOS as mentioned in the text, but instead presents alternative medicine (acupuncture, herbal medicine, etc.). 7. The authors claim that they added a sentence in the Introduction section stating that “knockdown of miRNA-21-5p leads to downregulation of PDCD4.” Unfortunately, this sentence is missing from the revised version of the text. This needs to be corrected. ****** 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 ******** [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. 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.0343735.r003
Revision 2
10 Jan 2026 Author Response Response to Reviewers Manuscript ID: [PONE-D-25-54068R1] Title: Knockdown of miR-21-5P targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates in sulin resistance in polycystic ovary syndrom Dear Dr. Jean-Marc A Lobaccaro and Reviewers, We would like to thank you for the opportunity to revise our manuscript. We are grateful to the Academic Editor and Reviewer for their constructive comments and positive evaluation of our work. These suggestions have significantly improved the quality and clarity of our manuscript. We have carefully addressed all the points raised by Reviewer. All changes in the revised manuscript have been highlighted using the "Track Changes" function. Below is a point-by-point response to the reviewer’s comments, with the modified text displayed in red. Response to Reviewer #1 Comment 1: The first paragraph of the Introduction still contains repetitions and syntactic heaviness that could be improved. Here is what could be suggested instead of the current third and fourth sentences... Response: Thank you for the suggestion. We have rewritten the sentences in the first paragraph of the Introduction exactly as recommended to improve clarity and flow. Revised text (paragraph 1, lines 4–8, introduction): PCOS is the most common cause of anovulatory infertility, with its pathogenesis driven by disruptions in sex hormone secretion, insulin resistance, and abnormal follicular development[4]. Insulin resistance represents a central pathological and clinical feature of PCOS [5-7].Consequently, establishing a reliable animal model is critical for progressing research in this field [8]. Comment 2: In the Materials and Methods paragraph “Primary Granulosa Cell Collection,” there are misorganized arguments that could lead to misunderstanding... Animals were anesthetized, not granulosa cells. Response: We apologize for the grammatical error. The sentence has been corrected to clarify that the rats were anesthetized before ovary collection, not the cells. Modified Text (Page 4, Primary granulosa cell collection�Materials and Methods): Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (3% Pelltobarbitalum Natricum, Merck, Germany, 0.03 ml/100 g), and then ovaries were collected to extract granulosa cells, following the protocol of a previous study [26]. Comment 3: In the “RNA Immunoprecipitation (RIP) Assay” paragraph, the references for the antibodies used in the experiment are missing. Response: We have added the detailed information (catalog number and brand) regarding the antibodies used in the RIP assay in the Materials and Methods section. Modified Text (Page 3,RNA Immunoprecipitation (RIP) Assay, Materials and Methods): For immunoprecipitation, 5 µg of anti-PDCD4 antibody (9535; Cell Signaling Technology, Danvers, MA, USA) or IgG control (2729; Cell Signaling Technology) was added to the lysate and incubated overnight at 4℃. Comment 4: In the “Protein Extraction and Western Blot Analysis” paragraph, the title suggests an expression analysis of PDCD4; however, the text does not provide any corresponding information (e.g., the antibody used and its reference). Response: We have supplemented the text with the detailed information for the PDCD4 antibody used for Western Blot analysis. Modified Text (Page 5, Materials and Methods): anti-PDCD4 antibody (1;2000, Cell Signaling Technology, Danvers, MA, USA) Comment 5: The name of the PDCD4 inhibitor, as well as the name of the company that supplied it, should be provided in the text the first time it is cited (Materials & Methods section). Response: The specific name of the PDCD4 inhibitor and its supplier have been provided in the Materials and Methods section upon its first citation. Modified Text (Page 6, PDCD4 Inhibitor Treatment in Primary Rat Ovarian Granulosa Cells (OGC),Materials and Methods): they were treated with a 10 mM PDCD4 inhibitor, PDCD4-IN-1 (CAS No. : 494763-64-3; MedChemExpress, Monmouth Junction, NJ, USA), was dissolved in DMSO and used to treat cells at the indicated concentrations. Comment 6: Reference 9 is not appropriate. It does not deal with Western medicine and/or therapies for treating PCOS as mentioned in the text, but instead presents alternative medicine (acupuncture, herbal medicine, etc.). Response: We agree with the reviewer. Reference 9 has been replaced with the 2023 International Evidence-based Guideline, which discusses standard Western medical therapies for PCOS. Modified Text (Page 3, Introduction): This disease is primarily treated with Western medicine drugs to promote ovulation or through surgery[9]. Updated Reference: Helena J T, Chau Thien T, Joop L, Anuja D, Lisa J M, Terhi T P, Michael F C, Jacky B, Leanne MR, Jacqueline AB et al: Recommendations from the 2023 International Evidence-based Guideline for the Assessment and Management of Polycystic Ovary Syndrome. Fertil Steril 2023, 120(4). Comment 7: The authors claim that they added a sentence in the Introduction section stating that “knockdown of miRNA-21-5p leads to downregulation of PDCD4.” Unfortunately, this sentence is missing from the revised version of the text. This needs to be corrected. Response: We sincerely apologize for this oversight in the previous version. The sentence regarding the relationship between miRNA-21-5p and PDCD4 has been correctly inserted into the Introduction section in this revised manuscript. Modified Text (Page 2, penultimate paragraph, Introduction): Given these multifaceted roles, PDCD4 represents a potential therapeutic target for PCOS. Importantly, recent studies have indicated a regulatory link between miRNAs and PDCD4. Specifically, it has been observed that knockdown of miRNA-21-5p leads to downregulation of PDCD4, suggesting a potential therapeutic axis[23]. We hope that these revisions meet the requirements for publication in PLOS ONE. Sincerely, Xiaohong Chen, MD Guangxi University of Traditional Chinese Medicine Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0343735.r004
11 Feb 2026 Decision Letter - Jean-Marc Lobaccaro, Editor Knockdown of miR-21-5P targets and regulates PDCD4-induced apoptosis in ovarian granulosa cells and ameliorates in sulin resistance in polycystic ovary syndrom PONE-D-25-54068R2 Dear Dr. chen, 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, Jean-Marc A Lobaccaro, PhD Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: The authors have adequately answered to the various comments. https://doi.org/10.1371/journal.pone.0343735.r005
Formally Accepted
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