PONE-D-25-09751SERPINH1 functions as a multifunctional regulator to promote the malignant progression of cervical cancerPLOS ONE

Dear Dr. Liu,

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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: Partly

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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: 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: The authors perform several bioinformatic analyses to show the importance of SERPINH1 in the promotion of cervical cancer malignancy. They also perform in vitro assays to support their findings. The manuscript is well written and has important findings. I consider it can be accepted after some minor comments are addressed.

Please specify how many times each experiment was repeated, either in the methods section or in the figure legends.

The subfigures of figure 4 are not correctly referred to in the text, please check.

The authors perform transfection experiments in SiHa and C33 cells, while they do shRNA experiments on HeLa cells. Why were these experiments performed on different cell ines? Please explain in the text.

The abstract states that This study systematically reveals the key role of SERPINH1 (Serpin Family H Member 1) as a hub regulator of malignant progression in cervical squamous cell carcinoma (CESC). However, HeLa cell line is an adenocarcinoma cell line and it is also used in this study. Please explain.

Section 6. Please define KIRP, LGG, MESO.

In the abstract, the authors mention the pathways uncovered by GSVA analysis (metabolism, netotic cell death and alkaliptosis). However, no experiments were perfomed to validate these findings. Although this is an important finding, this should not be mentioned in the abstract as one of the main findings in the study due to the lack of validation of the pathways.

As statistical analysis, the authors perform non-parametric analysis. Did the authors perform a normality test in their data in order to choose non-parametric tests? Please specify.

Reviewer #2: The manuscript presents a well-structured study with an appropriate methodological design. The topic is relevant, and the findings contribute to the understanding of SERPINH1 in cervical squamous cell carcinoma (CSCC). However, several issues should be addressed to strengthen the manuscript:

1. Lack of Supporting References in the Introduction:

The authors claim that the specific functions and regulatory mechanisms of SERPINH1 in cervical cancer remain poorly understood. This is an important point, but no references are provided to support this statement. Including relevant citations would substantiate the rationale for the study and contextualize its novelty.

2. Incomplete Literature Review in the Discussion:

While the discussion refers to some related studies, key prior work that could further support and enrich the interpretation of the findings is omitted. In particular, the study by Wang et al. (Front Genet., 2022; doi: 10.3389/fgene.2021.756094) offers valuable insights into the role of SERPINH1 across various cancers and should be included.

3. Low-Resolution Figures:

Figures 2, 3, and 6 are of suboptimal resolution, hindering proper evaluation of the results. The authors are encouraged to replace these with high-quality versions to ensure clarity and reproducibility.

4. Overstated Conclusions Regarding SERPINH1’s Role:

The manuscript proposes that SERPINH1 functions as a central regulator of malignant progression in CSCC via dual modulation of netotic cell death and alkaliptosis. However, the evidence presented does not conclusively support such a definitive role. The authors are advised to temper their conclusions and clearly distinguish between observed data and speculative interpretation.

Addressing these points will significantly enhance the clarity, scientific rigor, and impact of the manuscript.

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Reviewer #1: Yes:  Paola Maycotte

Reviewer #2: No

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<div>PONE-D-25-09751R1SERPINH1 functions as a multifunctional regulator to promote the malignant progression of cervical cancerPLOS ONE

Dear Dr. Liu,

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. The authors need to attended some reviewers comments before to accept the manuscript

Please submit your revised manuscript by Aug 16 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,

Victoria Pando-Robles, Ph.D.

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.

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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: (No Response)

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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: Partly

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

Reviewer #1: Yes

Reviewer #3: No

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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

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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: The authors have answered my previous comments and I have no further comments. I recommend the manuscript for publication.

Reviewer #3: In this research article, the authors aim to characterize the role of SERPINH1 in the progression of cervical squamous cell carcinoma (CESC), using several bioinformatics analyses and complemented by in vitro experiments. Through those analyses, it was identified that CC patients with high expression of SERPINH1 showed stromal remodeling, enhanced angiogenesis, and lipid metabolic alterations. However, the main results observed were that SERPINH1 is involved in proliferation, migration, and invasion pathways, which were identified in silico and corroborated with in vitro experiments.

This is an interesting and well-written research that is looking for the SERPINH1 role in CESC, but there are several points that need to be addressed.

General comment

It is not clear why the authors concluded that SERPINH1 is involved in the regulation of lipid metabolism. The results presented here showed that SERPINH1 is overexpressed, and several alterations could be related to this protein, but not necessarily in a direct effect. With the results obtained in silico, the authors cannot explain the mechanism through which SERPINH1 modulates this pathway. The authors are overestimating the results, and because of that, the discussion section needs to be modified.

In the in vitro assays, the authors need to better characterize the cell lines for SERPINH1 expression after being transduced or transfected to modulate SERPINH1 expression.

The cell lines used in the experiments were SiHa, which is a cervical cancer cell line that is transformed with HPV16; C33 is a cervical cancer cell line that does not contain HPV; and HeLa, which is an adenocarcinoma cell line transformed with HPV18. It will be important that the authors justify the use of different cell lines in the experiments and how the different genetic and viral backgrounds of the cell lines could affect the results.

An interesting result is presented in Fig. 6I, where the authors showed the regulation of the RNA expression levels of ITGA5, PLOD1, and ESM1 by SERPINH1. These results should be better discussed, as this is an event where SERPINH1 expression levels were directly involved and influenced the expression of these genes.

Specific points

Lines 141-145. Determine statistical differences between groups. Here, it is not clear which test was used, as for parametric data the t-test is used, but for non-parametric data the Mann-Whitney U is used. Please clarify and add this information to this section.

Line 224. Here it should be Fig. 4B and C

Line 226. The figure reference is wrong; this should be 4D and E

Line 228. Here again, the figure reference is wrong as it should be 4F-I

Lines 229-238. The authors suggested that SERPINH1 is overexpressed in CC and that the overexpression of this protein is related to the promotion of invasion and metastasis. To corroborate these findings that were identified from different databases, the researchers used cell lines that came from different cervical cancers. The researchers transduced SiHa and C33 to overexpress SERPINH1, but the basal protein levels seemed to be high. When the cell lines were transduced, no differences in SERPINH1 levels were observed (Fig. 4A). It would be desirable to carry out a densitometric analysis of this data and to have a quantitative analysis to identify the differences between the vector and the transduced SERPINH1.

On the other hand, the researchers used the HeLa cell line (not transduced), and it is clear from the Western blot that the basal levels of SERPINH1 were high in this cell line (Fig.5A). Why did the authors not use this cell line in all the experiments? How do the basal levels of SERPHIN1 affect or not the results observed?

Also, it would be ideal to determine the percentage of inhibition of SERPINH1 expression after siRNA transfection (Fig. 5A). This is important because siSERPINH1#1 seems to express lower levels of this protein than siSERPINH1#2, and this has different effects on the colony formation, migration, and invasion assays. Revise this and add this information to the text and in the corresponding figure to strengthen the analysis of the results.

Lines 231-238. In this part of the experiments to determine the role of SERPINH1 in proliferation, migration, and invasion by knocking down the gene, the researchers used the Hela cells. However, for the previous part of the experiments to show that overexpression stimulates these pathways, the cell lines used were SiHa and C33. There is no explanation for this change. The authors need to address the change of cell lines in the experiments and justify the use of the different cell lines.

Lines 302-306. The authors concluded that “SERPINH1 promotes the malignant progression of CESC by enhancing the proliferation and invasion capabilities of these cells”. These are the facts that were tested under in vitro conditions and that could be accepted to be associated with SERPINH1.

However, in this other part, “SERPINH1 in tumor malignancy through the regulation of lipid metabolism reprogramming, activation of glycan metabolic pathways, and modulation of novel cell death modalities such as NETosis and Alkaliptosis”, the authors are speculating too much about the role of SERPINH1 in regulating these pathways, as this analysis was made through analysis of patients bank genes. To confirm the role of SERPINH1 in these pathways, in vitro experiments are needed. The authors may construct an interaction diagram, which can better explain the interactions between these pathways and SERPINH1, and identify what needs to be done next.

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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:  Paola Maycotte

Reviewer #3: 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.

SERPINH1 functions as a multifunctional regulator to promote the malignant progression of cervical cancer

PONE-D-25-09751R2

Dear Dr. Liu,

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.

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Kind regards,

Victoria Pando-Robles, Ph.D.

Academic Editor

PLOS ONE

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