PONE-D-22-08405MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expressionPLOS ONE

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Additional Editor Comments:

According to all reviewers' comments, please address the concerns. The quality of this manuscript will be improved.

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: No

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: 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: No

Reviewer #2: Yes

Reviewer #3: 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: In this paper, the authors found that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. They further showed that this was correlated with suppression of EVI-1 signaling. The study was supported some solid data. However, few issues need to be addressed.

1. Please clarify how or where did you get the stem cells?

2. The materials and methods part is completely in a mess, please rewrite this part clearly.

3. In Fig 1B, how did the authors qualify the protein expression, please clarify in the methods part.

4. As showed in Fig 1D and E, the suppression effect was stronger at 72 hours instead of 48 hours, please check this result.

5. In Fig 2C and D, why the MTT assay did not inconsistent with flow analysis in Ad-CALR and Ad-CALR + miR-206 mimics group?

6. Could the authors provide representative images of transwell assay?

7. Many grammar mistakes and errors were noted. It is suggested that authors seek for professional editing service to improve the quality of language and the organization of the manuscript.

Reviewer #2: On the whole, it is a valuable and meaningful study, and the design in this study is logical. However, some points should be explained as following before publication.

1. Its research significance and potential application value should be discussed in the discussion

2. How to prove whether the transfection of MiR-206 mimics is successful?

3. What is the function of E-cadherin and N-cadherin in tumor progression? Why CALR exerted different effect on E-cadherin and N-cadherin?

4. Why Ad-CALR can not reverse the effect of MiR-206 on E-cadherin and N-cadherin?

5. Did the Ad-EVI-1 treatment affect the effect of miR-206 mimics on cytotoxic effect, E-cadherin and N-cadherin?

6. Please explained the principle and mechanism of miR-206 mimics, Ad-CALR and Ad-EVI-1 transfected into cells, what is different with plasmid transfection?

Reviewer #3: In this manuscript, Dapeng and co-authors describe the effect of miRNA-206 on the growth and migration of breast cancer cells and explore the underlying mechanism. Defining the role of miRNA-206 in breast cancer progression is useful to develop novel relevant prevention and/or therapy strategy . The authors did some work to validate their proposal. However, this study is not logically clear and some data is not convincing and need more detail work relating the underlying mechanism, particularly in relation to the relationship between miR-206, CALR and EVI-1 in their engaged signaling pathway.

Major comments

1. MM doesn’t clarify the origin of breast cancer stem cell, and more importantly these used stem cells should be fully/extensively characterized prior to functional assay, such as more stem cell markers besides CD44+/ CD24- should be employed to validate stem cells identity.

2. For the stem cells invasion/migration assay, it’s worth to include some representative images, that will be more visualized and convincing.

3. The final schematic is not consistent with the conclusion drawn in the text.

4. It is highly recommended to carefully go over the MM part to sort out some obvious incorrect description such as “transfected with 5ml Lipo 200”, repeated description, and incomplete method description.

5. Please include all the qPCR primers sequence used in this study.

6. The EVI-1 background introduction should be incorporated.

Minor comments

a. The cat # of Reagents/Cells/Instrument used in this study is missing.

b. Please number all the rows of the text part, which will be much easier to be located during the reviewing process.

c. The ms should be further polished regarding its writing in a professional way and tense using.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expression

PONE-D-22-08405R1

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.

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,

Ning Wei

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The current version of this manuscript is acceptable.

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: (No Response)

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 #2: (No Response)

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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: (No Response)

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: (No Response)

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: In this paper, the authors found that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. They further showed that this was correlated with suppression of EVI-1 signaling. The study was supported some solid data. And only a few issues need to be addressed.

1. Line 154-155, it should be actin instead of ACTN.

2. Please correct the uppercases and lowercases in the article.

3. For the Annexin V/PI staining, the authors can include Annexin V+ only part in Fig. E2.

Reviewer #2: (No Response)

Reviewer #3: Even the current version of the manuscript looks pretty decent, I would still strongly suggest performing extensive/full characterization of the stem cells in future study.

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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 #2: No

Reviewer #3: No

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