PONE-D-20-20790

MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation injury via modulating Notch1/PTEN/AKT signaling pathways in H9c2 cells

PLOS ONE

Dear Dr. Huang,

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Meijing Wang, MD

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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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: 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: 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: Kaempferide is a nature flavanol. Previously, other reports demonstrated that kaempferol improves I/R-induced cardiac dysfunction and myocardial injury via reducing myocardial infarct size, cardiomyocyte apoptosis, oxidative stress. In this study, Huang and Qi reported that kaempferol mediated protective effects in H9c2 cells against hypoxia/reoxygenation injury could be mediated by modulating of miR-21 expression to regulate Notch1/PTEN/AKT signaling pathways. The overall of the study is well designed and executed. Some major and minor issued need to be addressed are as below:

1. I/R injury induced cardiomyocyte drop-out is mostly associated with necrosis rather than in vitro H/R cell model associated cell apoptosis. Thus, using H9c2 cells in H/R model in this study may not fully reflect the true mechanism of kaempferide mediated in vivo cardiac-protective effects again I/R injury.

2. In Figure1 and Figure 2 D, the Flow cytometry analysis results of apoptosis (Fitc-Annexin positive) , late stage apoptosis (FITC-A and PI double positive) and necrosis (PI positive) is not consistent.

3. This study only examined overall Notch-1 transcription and protein level, but did not check the Notch intracellular domain (NICD). As NICD is a more direct reflection of Notch signaling activation, so it should be included in the WB analysis.

4. In methods section at Page 10, some of the words are written in Chinses which need to be replaced by English.

Reviewer #2: Ref: PONE-D-20-20790

In the present article entitled “MiR-21 mediates the protection of kaempferol against hypoxia/ reoxygenation injury via modulating Notch1/PTEN/AKT signaling pathways in H9c2 cells” Huang et al. have explored the involvement of miR-21 in the cardioprotective effect of kaempferol on hypoxia/reoxygenation (H/R)-induced H9c2 cell injury. Further they have tested the involvement of Notch/PTEN/Akt signaling pathway in such cardioprotective effect of kaempferol. The study design is simple and the results are well supported by the data. However, some points need to be addressed to make the study robust for the publication.

1. There are many grammatical errors in the manuscript. Especially the use of “the” needs to be checked throughout the manuscript.

2. Title is not clear. “protection of kaempferol against hypoxia/reoxygenation injury…” needs to be rephrased.

3. Authors have used “Kaempferide” or “kaempferol” or “kae” interchangeably. Please stick with one nomenclature.

4. Page 11, line 11 “Particularly, miR-21 is highly expressed in cardiomyocytes and has also been shown to regulate important processes of myocardial I/R injury”. Please provide some mechanistic information of how miR-21 is involved in myocardial I/R injury by citing other papers like Roy et al., PMID: 19147652; Gu et al., PMID: 25809568; Xu et al., PMID: 25159851.

5. Page 16, line 7, there is some text in different language. Please fix.

6. Statistical analysis. The samples size used in this paper is majorly 3. In this smaller number of samples, the normality check is not possible. Have the authors used non-parametric tests? Please mention.

7. Figure 1, Authors have selected 20 μM concentration of kae for all experiments. But in figure 1A and 1B, the concentration of kea used was 30 μM (without H/R, last bar). Authors should present data from 20 μM (without H/R) in some experiments.

8. Figure 1D: The bar graph (H/R group) presented does not match with the scatter plot data shown in Figure 1C.

9. Page 20, line 10, Figures 1F and 1G are not cited.

10. Page 20, line 13, “and apoptosis [37]. The present further found that following kae pretreatment”. It should be “The present article further…...”

11. Figure 2D: The bar graph (H/R and Kae+H/R+miR-21 I groups) presented does not match with the scatter plot data.

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Reviewer #1: No

Reviewer #2: No

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MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

PONE-D-20-20790R1

Dear Dr. Huang,

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,

Meijing Wang, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

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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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: (No Response)

Reviewer #2: (No Response)

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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