PONE-D-25-10451Downregulation of  OIP5-AS1 Inhibits Apoptosis in Myocardial Ischemia/Reperfusion Injury via Modulating the MiR-145-5p/ROCK1 AxisPLOS ONE

Dear Dr. Zhang,

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 check the reviewers' comments. Ensure that all figures are appropriately cited and that the methods are described in sufficient detail for reproducibility. Include catalog IDs for all products used, as well as the sequences for the miR-145-5p mimic. Specify which method was used for the 2^-ΔΔCq calculation (Livak, Pfaffl, or another approach) and provide the corresponding citation. Clearly describe the procedure for adaptor ligation during miRNA-to-cDNA conversion. Define all abbreviations upon their first mention and thoroughly review the English grammar throughout the document. Include melting curves to confirm the specific amplification of all target regions, including miR-145-5p. Replace bar plots with box plots or violin plots, supplemented by point or jitter plots to display all biological replicates, particularly for expression analyses, as this provides a more transparent representation of the results.

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PLOS ONE

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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

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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: Yang and his colleagues observed that the expression of the long non-coding RNA (lncRNA) OIP5-AS1 and apoptosis were increased in myocardial ischemia/reperfusion (I/R) injury. Based on their previous study, they further confirmed that the expression of miR-145-5p was increased, while its target, ROCK1, was decreased. They then demonstrated that reducing OIP5-AS1 expression led to a decrease in apoptosis and experimentally validated the relationship between OIP5-AS1 and miR-145-5p.

The paper presents a very straightforward mechanism, and the experimental evidence supporting each claim is convincing.

However, to strengthen the authors' argument, I suggest including additional experiments to clarify the relationship between ROCK1 and OIP5-AS1:

1. Does overexpression of ROCK1 in the context of reduced OIP5-AS1 modulate the level of apoptosis?

2. How does apoptosis change when anti-miR-145-5p is introduced in a setting where OIP5-AS1 is downregulated?

Reviewer #2: Thank you for the opportunity to review your manuscript titled "Downregulation of OIP5-AS1 Inhibits Apoptosis in Myocardial Ischemia/Reperfusion Injury via Modulating the MiR-145-5p/ROCK1 Axis." Your study addresses an important and relevant area of research with potential implications for novel therapeutic strategies targeting myocardial ischemia/reperfusion injury. The exploration of OIP5-AS1's interaction with miR-145-5p and its downstream impact on ROCK1 is promising and could significantly contribute to the existing literature.

However, the methodology section needs substantial improvement. The descriptions provided are too simplified and lack critical details necessary for replication. Specifically, information regarding the exact concentrations of siRNAs, miRNA mimics, and inhibitors utilized in the experiments is missing. Additionally, you should justify the choice of Lipofectamine 2000 over the more RNA-specific reagent Lipofectamine RNAiMAX. Clarification about whether optimization experiments using "killer" siRNA were conducted would also strengthen your methodological transparency. Including control experiments with Lipofectamine alone would clarify if observed effects are due specifically to the treatments or influenced by reagent toxicity. Also, crucial details such as the quantity of RNA used in qPCR reactions, conditions for replicates (technical or biological), the acceptable Ct variation between replicates, and explicitly mentioning the normalization gene used (presumably snU6) must be clearly stated.

In your results section, findings are presented clearly and accompanied by suitable statistical analyses. Nevertheless, minor typographical errors were identified—for instance, "Conrtol" should be corrected to "Control" in Figure 2. Moreover, please clarify whether control groups containing only Lipofectamine (without siRNAs or mimics) were included to exclude nonspecific reagent effects.

Your discussion section is somewhat superficial and would greatly benefit from a more thorough examination of the study's limitations. It is essential to explicitly address limitations, such as small sample sizes, potential off-target effects of transfection agents, and methodological constraints inherent to the model systems used. Additionally, clearly stating future research directions would significantly strengthen this section. For example, suggesting studies that could confirm your findings in larger or more clinically relevant contexts would substantially enhance the manuscript’s scientific and clinical significance.

Furthermore, you should provide clear steps or recommendations to explore the clinical applicability of your findings. For instance, how might the OIP5-AS1 and miR-145-5p/ROCK1 axis modulation translate into clinical interventions or therapeutic approaches?

Lastly, a minor typographical and grammatical revision is necessary throughout the manuscript to maintain professional standards, such as correcting the spelling of "Conrtol" to "Control" in Figure 2. A professional language review is recommended prior to resubmission.

In conclusion, the manuscript presents valuable and promising insights but requires substantial methodological clarifications, deeper acknowledgment of limitations, and a more comprehensive discussion before consideration for acceptance.

Reviewer #3: The authors showed that a long non-coding RNA OIP5-AS1 is upregulated upon ischemia/reperfusion (I/R)-induced apoptosis, and can act like a sponge for miR-145-5p and release the miR-145-5p target (ROCK1) expression. Both in vitro (culture cells) and in vivo models were used.

I could read until Figure 2 end, but started to fell unconfortable for reading the result section afterwards. This is because Figures are not cited correctly in the Result section of maintext and the writing is poor especially in the latter half.

[Methods section]

“miR-145 mimics and inhibitors” information was not shown.

[Result section]

In the paragraph “MiR-145-5p directly targeted by OIP5-AS1 and expressed at low levels in I/R”, Figure 3B is first cited, 3A cannot be found. Neither to 3C 3D 3F.

“Furthermore, silencing of OIP5-AS1 in I/R significantly upregulated miR-145-5p

expression (Fig. 2E).” – Figure 2E is not showing this evidence.

“These results indicated that miR-145-5p was downregulated in

I/R and was a target of OIP5-AS1.” – Better rephrase. Generally, a transcript is a target of miRNA.

“Western blot analysis

and RT-qPCR were performed to investigate ROCK1 expression. Results showed significant upregulation of ROCK1 in I/R cell models compared with controls (Fig.1F).” – Fig.1F is RNA data. No WB data was shown.

“The luciferase report assay demonstrated that miR-145-5p mimics substantially attenuated the luciferase activity of ROCK1-WT, while ROCK1-MUT showed no changes in response to miR-145-5p elevation (Fig. 3F).” – Need rephrase. More correctly, "no change after addition of miR-145-5p mimics.” It is very confusing that this figure is placed as Fig 3F.

“ROCK1 expression was reduced in I/R by OIP5-AS1 deficiency while miR-145-5p was highly

expressed (Fig. 2F and 2G).” – These data are showing ROCK1 RNA and protein levels. but not miR-145-5p level.

“Altogether, OIP5-AS1 behaved as a ceRNA against miR-145-5p, resulting in ROCK1 upregulation.” – Cannot give such strong conclusion from these data. Just “suggest”.

“ceRNA” appears here for the first time without explanation. What is it.

“Figure 5” appears in the last paragraph, without any explanation of individual panels (5A~E). Very poor and unfriendly writing.

[Discussion section]

“OIP5-AS1. OIP5-AS1 was found to inhibit the protective function of miR-145-5p, and it was confirmed to target miR-145-5p.” – “confirmed” sounds too strong compared to the presented data.

The authors can try OIP5-AS1 overexpression experiment in the absence of IR treatment in vitro and in vivo. “a transcript is targeting a miRNA” is confusing, and better be rephrased, like “ OIP-AS1 can sequester miR-145-5p” or “OIP-AS1 has the target site of miR-145-5p”

“This further supports the relationship between OIP5-AS1and miR-145-5p.” – should explain more about the “relationship” in this sentence.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-25-10451R1Downregulation of  OIP5-AS1 Inhibits Apoptosis in Myocardial Ischemia/Reperfusion Injury via Modulating the MiR-145-5p/ROCK1 AxisPLOS ONE

Dear Dr. Zhang,

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 verify minor language and typo corrections suggested for reviewers, and run a complete proofreading of the manuscript.

Please submit your revised manuscript by Jun 14 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Alexis G. Murillo Carrasco

Academic Editor

PLOS ONE

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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.

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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

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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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

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: Yes

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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: Thank you for conducting all the experiments I suggested and for including the corresponding results.

Reviewer #2: I have carefully evaluated the revised manuscript entitled "Downregulation of OIP5-AS1 Inhibits Apoptosis in Myocardial Ischemia/Reperfusion Injury via Modulating the MiR-145-5p/ROCK1 Axis."

I appreciate the authors' efforts to thoroughly address all the concerns raised in the initial review. The manuscript has undergone significant improvements, particularly in the following aspects:

Methodology: The authors have now provided detailed descriptions of the experimental procedures, including the concentrations of siRNAs, mimics, and inhibitors, the rationale for choosing Lipofectamine 2000, and optimization details. The methodology is now sufficiently transparent to allow reproducibility.

Controls: Appropriate negative controls were included and clearly described, addressing concerns about potential nonspecific effects of transfection reagents.

Results Presentation: Minor typographical errors, such as the correction of "Conrtol" to "Control," have been rectified.

Discussion and Limitations: The authors have expanded the discussion significantly, explicitly acknowledging key limitations and proposing valuable future research directions, which strengthen the manuscript's scientific rigor and translational relevance.

Language and Style: The manuscript's language has been improved considerably. Although minor editorial polishing could further enhance clarity, it does not hinder comprehension or scientific value.

Given the authors' careful and thoughtful revisions, I find that all my initial concerns have been satisfactorily addressed.

Therefore, I recommend the manuscript for acceptance after minor language editing (optional proofreading).

Congratulations to the authors for their excellent work and for the significant improvements made to the manuscript.

Reviewer #3: Many points were addressed by the authors.

I just give some points that would need correction before publication.

Table 1

si-OIP5-AS1 and miR mimic‑NC contain "T" in their sequence. If true, why control does not have them.

"Concentration" cannot be understood. Final concentration in culture?

Table 2

Oligos for miRNA would not be correct. Probably the reverse one is an adaptor for RT. Universal Rv primer should be used here.

I have no idea if "miR, microRNA; ROCK1, Rho-associated coiled-coil-containing kinase 1" included in Table 2 is needed in this position.

Line 246

Sentence stopped in the middle. "compared" to what.

Line 268

I didn't get what "pivoral" indicates here.

Line 275

Figure 5 and "8"

8A and 8B are not cited, although authors stated all Figures are to be correctly cited.

Figure legends for Fig1~9 were provided as supporting information.

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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: No

Reviewer #3: No

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Downregulation of  OIP5-AS1 Inhibits Apoptosis in Myocardial Ischemia/Reperfusion Injury via Modulating the MiR-145-5p/ROCK1 Axis

PONE-D-25-10451R2

Dear Dr. Zhang,

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.

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Kind regards,

Alexis G. Murillo Carrasco

Academic Editor

PLOS ONE

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