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Dear Dr Sun,

Thank you very much for submitting your Research Article entitled 'DNA methylation-mediated repression of exosomal miR-652-5p expression promotes oesophageal squamous cell carcinoma aggressiveness by targeting PARG and VEGF pathways' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations, and we would then be pleased to consider it again for possible acceptance. Your revisions should address the specific points made by each reviewer.

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Yours sincerely,

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Associate Editor

PLOS Genetics

David Kwiatkowski

Section Editor: Cancer Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Overview

The manuscript is about a research on miR-652-5p in esophageal cancer, particularly ESCC. With human ESCC tissues, the authors found miR-652-5p is downregulated in a number of ESCC tissues. Importantly, the downregulation of miR-652-5p is correlated with the prognosis of ESCC patients. The down regulation of miR-652-5p in the blood samples of ESCC indicated miR-652-5p is a potential blood biomarker for the prognosis of ESCC.

From the experimental perspective, I think the manuscript provided solid in vitro and in vivo evidences to support the author’s conclusion that repression of exosomal miR-652-5p expression promotes the aggressiveness of ESCC. The authors also provided adequate molecular mechanistic work to elucidate the molecular pathways that are altered in miR-652-5p downregulated ESCC, and epigenetic evidences for its downregulation. Thus, regarding the general significance of the findings, and the overall quality of the experiments of the manuscript, I think the manuscript meets the standard and scope of Plos Genetics, and is of general interests to the readers of the journal. I recommend for its acceptance and publication by Plos Genetics.

Reviewer #2: In this article, Gao et al. have studied the biological functions, clinical significance and therapeutic implications of miR-652-5p in ESCC. They found that down-regulated expression of miR-652-5p by hypermethylation of its promoter may contribute to the progression of ESCC. And miR-652-5p might be considered as a potentially effective biomarker. This study is well organized and has certain innovation. However, there are some minor issues that should be concerned.

1. In the "Materials and methods" section, the process of "In situ hybridization" should be described in detail.

2. In the "Evaluation of immunohistochemical staining" section, detailed information of antibody and the histological scoring method should be described.

3. For some the figures, the authors did not show clearly how many experiments they repeat.

4. How to choose the candidate genes in Figure 5a and 5b? The authors should give more details.

5. There is no mention of the amount of miR-652-5p that was transfected into the cells for any of the experiments. This is a critical parameter as excess miRNA can lead to off target effects through binding to low-affinity targets and/or sequestering Ago from other miRNAs. The concentration used in these experiments needs to be defined at least in the methods; however, inclusion in the figure legends would also be helpful.

6. For colony formation assays that were performed over 10 days, there is a concern that the miRNA would be diluted out due to replication. A better approach would be to use a lentiviral miRNA expression vector to achieve constitutive expression.

Reviewer #3: The research is good and meanful to molecular mechanism of ESCC. While it would be better if the manuscript would be thoroughly revised in its english sentences.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Dear Dr Sun,

We are pleased to inform you that your manuscript entitled "DNA methylation-mediated repression of exosomal miR-652-5p expression promotes oesophageal squamous cell carcinoma aggressiveness by targeting PARG and VEGF pathways" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional accept, but your manuscript will not be scheduled for publication until the required changes have been made.

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If you have a press-related query, or would like to know about one way to make your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Peter Hammerman, MD, PhD

Associate Editor

PLOS Genetics

David Kwiatkowski

Section Editor: Cancer Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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Data Deposition

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