PONE-D-22-16558

Hsa-miR-22-3p inhibits liver cancer cell EMT and cell migration/ invasion by indirectly regulating SPRY2

PLOS ONE

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, follow the suggestions form the reviewers to improve the manuscript. In particular, include a rationale to studying miR-22-3 in this context. Also, add more detailed information to the materials and methods: One of the PLOS ONE publication criteria is that “Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.” This implies that other researcher can reproduce the experiments described. In its current form, this is not the case as pointed out by reviewer 1. In addition, you should correct the figures and change from bar or column graph to box or dots plots, so the readers can identify the data points.

I also request that you proof-read your manuscript  and expand the discussion, especially as is difficult to evaluate whether the described "axis" is relevant in vivo and what the normal role of miR-22 in the liver might be. A context and possibly explaination should be added to why other studies using KO cells (e.g., PMID: 25323629) show more or less exactly the opposite. It is essential to include how a microRNA such as miR-22 can have fundamentally different effects on the very same genes in two different settings. 

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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: Yes

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

Reviewer #1: No

Reviewer #2: 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

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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: Yes

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: Summary: This paper attempts to elucidate the mechanism and function of miR-22 in liver cancer. While this miRNA has different known functions depending on the cancer, several studies have shown that in hepatocellular carcinoma (HCC) there is a marked decrease in miR-22, which suggests that miR-22 can function as a tumor suppressor. In this paper, the authors link miR-21-3p with direct inhibition of CBL, which stabilizes SPRY2 and prevents the epithelial mesenchymal transition (EMT) in HCC cells. Please address the points below.

Major Points:

• On page 4, in the introduction, there is no differentiation between miR-22-5p versus miR-22-3p. This is a key element that should be explained in the introduction, especially since the 3p and 5p functions of miRNAs are still being examined. The rationale is also missing as to how they choose miR-22-3p as their target of interest.

• Make sure that the verb tense is consistent throughout the paper. Most of the paper is in past tense, but there are present tense verbs present in several pages (4 and 12).

• In page 4, the authors mention senescence-like phenotypes in HCC cells, but how does that connect to the anti-tumor role miR-22 has in HCC?

• In the introduction on page 5, the paper discusses the MAPK signaling pathway briefly, but it should be made clear what the connection between ERK and the MAPK signaling pathway are. Also, how is SPRY2 a modulator of this pathway? At what step? Is CBL also part of the MAPK signaling pathway?

• For the migration and invasion assays, the quantification was done manually by counting cells in the images. Is there an alternative method of quantification such as ImageJ or a cell counting software that could be more objective?

• In figure 1D, 1E, and 1G, the y-axes are missing units and titles.

• The migration and invasion images in figures 1 and 3 are all missing scale bars. Also, arrows may be useful for pointing out significant movement or lack thereof of the cells.

• In figure 1, how were the cells quantified? Were they counted as groups or individual cells per frame?

• In figure 1, why is Huh7 not included in the cell migration and invasion assays? Did they knockdown and/or overexpress miR-22-3p in these cells as well?

• On page 16, it is not clear how CBL was re-expressed in miR-22-3p overexpression cells was done? Lenti-viral vector? Plasmid?

• In figure 4, there is no statistical analyses for the western blot.

• For figure 4D, the authors could also show, visually, how knocking out or inhibiting miR-22 would have the opposite effect.

• Limitations and further applications need to be discussed in the conclusion.

• They mention that miR-22 can be a potential biomarker, but are there other applications? Clinical or otherwise?

Minor Points:

• In the beginning of the abstract, there could be more of a brief explanation of how miR-22 can function as a tumor suppressor or promoter, like through what mechanisms?

• Several times in the paper, the “r” in miR-22 is lowercase when it should be uppercase. Or the “m” is uppercase when it should be lowercase. This is notable in pages 4, 5, and 12.

• At the top of page 4, it should be “miR-22 is one such dual-functioning miRNA in cancer”.

• A brief definition of ubiquination somewhere in the introduction would be helpful for the readers.

• In the materials and methods section, there is no catalog number for DMEM (high glucose).

• The cells are not cited properly in the cell culture section of the materials and methods.

• On page 11, “To explore the existence of the miR-22/CBL/SPRY2 axis and the significance of this axis for the miR-22 function in HCC cell behavior, the effect of miR-22-3p on HCC EMT, migration/invasion, and CSC features were first studied.”

• On page 15, qPCR not QPCR.

• In figure 3, some of the graphs do not have standard deviations shown or p-values (3A, B, C, D).

• For the Western blot, the abbreviation for kilodalton is kDa, not kd.

Reviewer #2: The manuscript describes the impact of a well studied microRNA, miR-22, on the biology of two liver cancer cel lines. Although well established that this microRNA has inhibitory effects on liver cancer cells, the authors add some interesting data on how miR-22 may exert its tumor suppression.

The data appear robust, the line of thought is clear, and the presentation of the data convincing.

Although not much new is added to our understanding of microRNA biology and HCC, this may be true to many similar manuscripts. It is difficult to evaluate whether the described "axis" is relevant in vivo and what the normal role of miR-22 in the liver might be. Furthermore, cleaner experiments in other tumor types using KO cells (e.g., PMID: 25323629) show more or less exactly the opposite.

It would be nice to hear in the discussion how a microRNA such as miR-22 can have fundamentally different effects on the very same genes in two different settings. Overall, a more critical discussion may be helpful.

Furthermore, there are too many bar graphs. bar graphs hide data and therefore box plots are preferable and requested.

In summary: better plots, better discussion.

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Reviewer #1: No

Reviewer #2: No

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Hsa-miR-22-3p inhibits liver cancer cell EMT and cell migration/ invasion by indirectly regulating SPRY2

PONE-D-22-16558R1

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. Please note that I have acted as a reviewer for this manuscript, and you will find my comments below, under Reviewer 3.

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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Kind regards,

Atsushi Hosui, PhD, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All criticisms and suggestions written by reviewers were responded and addressed. This manuscript is novel and worth reading.

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 #3: 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

Reviewer #3: Yes

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

Reviewer #1: N/A

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 #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 #3: 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: The authors have addressed all the questions sufficiently. I am satisfied with the responses and revision.

Reviewer #3: All comment by reviewers have been addressed. This manuscript has been improved and becomes worth reading for readers.

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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: No

Reviewer #3: No

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