PONE-D-23-00076MicroRNA-708 emerges as a potential candidate to target undruggable NRASPLOS ONE

Dear Dr. LIN,

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. Your manuscript has been read by two well qualified reviewers. As you can see from the reviewers' comments below, the reviewers raised significant concerns on the quality of the manuscript. The manuscript would require revisions and new experiments to strengthen the conclusions. In addition, the reviewers also showed concerns on the statistics and English problems. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

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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: No

Reviewer #2: Yes

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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: The present manuscript describes a report using miRNA-708 that targets the NRAS gene to develop a miRNA-based management of NRAS mutation-driven cancers. They show that NRAS is a direct target of miRNA-708 which reduce NRAS protein levels in cell lines with NRAS mutation (melanoma, leukemia, and lung cancer), resulting in suppressed cell proliferation, anchorage independent growth, and promotion of reactive oxygen species-induced apoptosis. Its results also shown that the activities of NRAS-downstream effectors, PI3KAKT-mTOR or RAF-MEK-ERK signaling pathway, were affected in miR-708 over-expressing cells. Moreover, they shown that cell proliferation was not disturbed by miRNA-708 in cell lines carrying wild-type NRAS. They conclude that their results unveil the therapeutic potential of miRNA-708 in NRAS mutation-driven cancers as a precision medicine in cancer treatment.

In my opinion the study is well conducted and the results support the claimed conclusions. However, I believe that the manuscript can be improved resolving the following concerns.

Made clear in the whole manuscript when the symbols are referred to proteins or its genes…

Write properly the symbols of genes described (italic). Ej. RAS, NRAS and HRAS in the whole document when refers to gene names…

The last sentence in Survival Correlation Analysis in Material & Methods section is incomplete (page 13, line 249).

Fig 5 A, properly indicate the legend…

Respect to the results of the miRNA-708 over-expression on the activities of NRAS-downstream effectors, PI3KAKT-mTOR or RAF-MEK-ERK signaling pathway, a paragraph with information about other miRNA-708 targets that could explain the results on the activity of the ERK in SK-MEL-2 and TPH-1 cell lines should be included.

The survival-miRNA-708 expression correlation study results should be included in the discussion section…

I recommend the review of the manuscript by a native English language speaker

Reviewer #2: 1. Based on the experimental evidence provided by the authors, it is not clear whether NRAS is a direct or indirect target of mir-708. Is there any experimental evidence of NRAS and mir-708 interaction?

2. The authors should have included another control cell line with normal NRAS expression in order to draw the conclusion that the overexpression of miRNA-708 decreased the NRAS protein levels in the cell lines with NRAS mutation.

3. Overexpression of mir-708 only has a considerable anti-tumour impact on SK-ME-2 cells compared to the other two cell lines. Not all of the metrics taken into consideration for THP-1 and H1299 cells show a significant anti-tumour impact. In the result section (lines 308-310), the assertion drawn from the experiments does not indicate the same.

4. The lack of a robust anti-tumour effect from NRAS knockdown in SK-ME-2 raises the question of whether it is a potent therapeutic target.

5. What could be the plausible explanation for the differential response of mir-708 overexpression towards downstream effectors of NRAS?

6. In the Kaplan-Meier plot, the P value is quite high for the data to be considered significant. Thereby the assertions drawn may not hold true.

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Reviewer #1: Yes: Homero Reyes de la Cruz

Reviewer #2: No

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MicroRNA-708 emerges as a potential candidate to target undruggable NRAS

PONE-D-23-00076R1

Dear Dr. LIN,

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.

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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,

Ming Tan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript is acceptable now. One of the reviewers mentioned a few minor editorial issues. I think these can be corrected during copy editing or proofreading.

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

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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

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

Reviewer #1: 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

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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

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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: I believe that the authors took into account all the observations made to the manuscript and from my point of view they have made the appropriate corrections and added the suggestions, so I believe that the manuscript has improved considerably and can now be accepted for publication. However, the manuscript still has minimal editing errors that can be corrected. E.j., Page 4, line 65; “RAS” should be in normal (not cursive) letters; a space is missing, line 180; write properly 4oC, line 197…

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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: Yes: Reyes de la Cruz, Homero

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