PONE-D-24-47234Artesunate Regulates the Malignant Progression of Breast Cancer Cells via the lncRNA TUG1/miR-145-5p/HOXA5 AxisPLOS ONE

Dear Dr. Yang,

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 submit your revised manuscript by Apr 04 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Manikkam Rajalakshmi

Academic Editor

PLOS ONE

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 [This work was supported by the Training Project of Clinical Medical Professionals

(2023), ZF2023065, provided by the government of Hebei Province.]. 

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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: 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: Review of Manuscript PONE-D-24-47234: Artesunate Regulates the Malignant Progression of Breast Cancer Cells via the lncRNA TUG1/miR-145-5p/HOXA5 Axis

Overall Assessment:

This manuscript presents a comprehensive study on the potential anti-cancer effects of artesunate on breast cancer cells, focusing on the lncRNA TUG1/miR-145-5p/HOXA5 axis. The study employs a variety of in vitro assays to explore the proposed pathway, providing valuable insights into the molecular mechanisms underlying artesunate's action. However, there are several areas that require improvement and clarification to enhance the manuscript's quality and impact.

Major Comments:

1. Background and Context:

- The introduction effectively sets the stage by highlighting the challenges in breast cancer treatment and the potential of traditional Chinese medicine, specifically artesunate. However, it would be beneficial to include more recent references to underscore the current state of research in this area.

2. Methodology:

- The methods section is detailed and provides a clear understanding of the experimental procedures. However, it would be helpful to include a flowchart or diagram to illustrate the experimental design and timeline, especially for the drug stimulation and plasmid transfection experiments.

- The rationale for choosing specific concentrations of artesunate (30μmol/L, 120μmol/L, and 480μmol/L) should be explained. Are these concentrations based on previous studies or preliminary experiments?

3. Results:

- The results section is well-organized and presents a clear narrative of the findings. However, some figures and tables are not provided in the manuscript, making it difficult to fully assess the data. Ensure that all relevant figures and tables are included.

- The discussion of the results could be strengthened by comparing the findings with existing literature on artesunate and its effects on cancer cells.

4. Discussion and Conclusions:

- The discussion effectively summarizes the key findings and their implications. However, it would be beneficial to explore the potential clinical applications of artesunate in breast cancer treatment more thoroughly.

- The conclusions are clear and concise, but they could be further supported by highlighting the study's limitations and suggesting avenues for future research.

5. Ethics Statement:

- The ethics statement is brief and does not provide sufficient detail. Given that the study involves cell lines, it would be appropriate to include a statement confirming that the cell lines were obtained from a reputable source and that appropriate ethical guidelines were followed.

6. Data Availability:

- The data availability statement is clear and indicates that all data are fully available without restriction. However, it would be helpful to specify where the data can be accessed, such as a public repository or as supplementary materials.

Minor Comments:

1. Formatting and Style:

- There are inconsistencies in formatting and style throughout the manuscript. Ensure that the manuscript adheres to the PLOS ONE submission guidelines.

2. References:

- Some references are missing or incorrectly formatted. Ensure that all references are complete and correctly formatted according to the PLOS ONE style guide.

3. Typos and Grammar:

- There are several typos and grammatical errors throughout the manuscript. A thorough proofread is recommended to improve clarity and readability.

Major Concerns

Study Design

- The concentration range for artesunate (30-480 μmol/L) is quite wide and requires justification based on physiologically achievable levels .

- Control experiments using non-malignant breast cells (MCF10A) should be more extensively described .

Technical Issues

- The Western blot methodology lacks details about loading controls and quantification methods .

- The plasmid transfection efficiency validation data is not presented .

Data Presentation

- Statistical analyses methods are not clearly described .

- Some experimental replicates numbers are not specified .

Recommendations

1. Include dose-response curves for artesunate treatment

2. Add detailed statistical analysis methods

3. Provide quantification for Western blot data

4. Validate key findings using additional breast cancer cell lines

5. Include transfection efficiency data

Reviewer #2: 1. Justify dose selection based on pharmacokinetic data or previous in vivo studies to ensure translational relevance.

2. Include details on assay validation and reproducibility to strengthen methodological rigor.

3. Conduct rescue experiments (e.g., overexpression or inhibition of miR-145-5p/HOXA5) to confirm the pathway’s role in artesunate’s effects.

4. Include in vivo experiments (e.g., xenograft mouse models) to validate the translational potential of artesunate.

5. Add an ethics statement regarding the sourcing and use of cell lines, as per PLOS ONE guidelines.

6. The introduction contextualizes artesunate well but could benefit from more discussion on its pharmacokinetics and potential side effects. I recommend that authors include a brief discussion on artesunate’s pharmacokinetics, toxicity, and challenges in clinical application.

7. The figures could be clearer, kindly provide clearer versions of them.

8. Elaborate on limitations such as the in vitro nature of the study, lack of long-term toxicity data, and absence of patient-derived models in the limitations section.

9. Reframe the conclusion to emphasize the need for further in vivo and clinical studies.

10. Including computational modelling (e.g., molecular docking) to explore artesunate’s interaction with lncRNA TUG1 and related targets will be advantageous.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-47234R1Artesunate Regulates the Malignant Progression of Breast Cancer Cells via the lncRNA TUG1/miR-145-5p/HOXA5 AxisPLOS ONE

Dear Dr. Yang,

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 submit your revised manuscript by Jun 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Manikkam Rajalakshmi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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: Partly

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: 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: 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: 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: 1. Lack of In Vivo Validation

- Critical Gap: The study relies entirely on in vitro models (MCF7, HCC1395). While these are standard, the absence of in vivo data (e.g., xenograft models, PDX) severely limits translational relevance.

- Specific Concerns:

- ART’s efficacy in suppressing tumor growth, metastasis, and toxicity in living systems remains unverified.

- No pharmacokinetic/pharmacodynamic (PK/PD) data to confirm whether the proposed TUG1/miR-145-5p/HOXA5 axis operates in vivo .

- Recommendations:

- Include xenograft experiments using ART-treated mice to validate tumor suppression and pathway modulation.

- Measure ART and metabolite levels (e.g., DHA) in serum/tissue to correlate dosing with effects.

2. Clinically Irrelevant Dosing

- Critical Issue: ART concentrations (30–480 μM) far exceed pharmacologically achievable levels in humans. Typical plasma concentrations after standard malaria dosing (2–4 mg/kg) are ~1–10 μM.

- Specific Concerns:

- High doses (e.g., 480 μM) risk off-target toxicity and may not reflect therapeutic windows.

- No justification for dose selection or comparison to prior preclinical/clinical studies.

- Recommendations:

- Justify dose ranges using PK data (e.g., cite studies where ART inhibited cancer cells at ≤10 μM).

- Test lower, clinically relevant doses (1–20 μM) to confirm pathway-specific effects.

- Discuss potential toxicity (e.g., hepatotoxicity, bone marrow suppression) at high doses.

3. Narrow Mechanistic Focus

- Critical Oversight: The study focuses solely on the TUG1/miR-145-5p/HOXA5 axis, ignoring ART’s well-documented roles in ferroptosis, autophagy, and ROS generation.

- Specific Concerns:

- No experiments to rule out contributions from other pathways (e.g., GPX4 for ferroptosis, LC3B for autophagy).

- Overlooked crosstalk between WNT/β-catenin and other oncogenic pathways (e.g., STAT3, AKT).

- Recommendations:

- Perform RNA-seq/proteomics to identify additional ART-regulated pathways.

- Inhibit ferroptosis (e.g., ferrostatin-1) or autophagy (e.g., chloroquine) to test if ART’s effects persist.

4. Limited Model Diversity

- Issue: Only two breast cancer cell lines (MCF7, HCC1395) were tested, both luminal/subtypes. Triple-negative (TNBC) or HER2+ models are absent.

- Recommendation: Include TNBC lines (e.g., MDA-MB-231) to assess broader applicability.

5. Data Transparency

- Issue: Uncropped Western blot images and raw qPCR data are not clearly accessible via the Dryad link.

- Recommendation: Provide annotated, full-length blot images and raw Ct values in SI.

Critical Revisions Required

1. Prioritize In Vivo Experiments: At minimum, include pilot xenograft data to support translational claims.

2. Re-evaluate Dosing Strategy: Align ART concentrations with clinically achievable levels and provide pharmacokinetic rationale.

3. Broaden Mechanistic Scope: Address alternative pathways (e.g., ferroptosis) to confirm the TUG1 axis is the primary driver.

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: Yes:  Ahmed I. Abd El Maksoud

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-24-47234R2Artesunate Regulates Malignant Progression of Breast Cancer Cells via

lncRNA TUG1/miR-145-5p/HOXA5 AxisPLOS ONE

Dear Dr. Yang,

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.

==============================Note from the Editorial Office: We note that you have not yet included the source and authentication of the artesunate product used in your study. This would typically involve a chemical characterization of the product. Please revise your manuscript to address this query, or explain why this was not possible. Thank you.

==============================

Please submit your revised manuscript by Aug 04 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Sarah Jose, Ph.D.

Staff Editor

PLOS ONE

on behalf of

Manikkam Rajalakshmi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Artesunate Regulates Malignant Progression of Breast Cancer Cells via

lncRNA TUG1/miR-145-5p/HOXA5 Axis

PONE-D-24-47234R3

Dear Dr. Chao Yang 

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,

Manikkam Rajalakshmi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: