Submit to this Journal Review for this Journal Propose a Special Issue

Article Menu

Share Help Cite Discuss in SciProfiles

Open AccessArticle

Peer-Review Record

Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of MicroRNAs

Biomolecules 2026, 16(3), 366; https://doi.org/10.3390/biom16030366

by Nouman Amjad^1,2, Muhammad Majid¹

, Zhaojian Sun¹

, Rajesh Basnet¹, Kashaf Rasool³, Linping Wu¹

and Zhiyuan Li^1,4,*

Reviewer 1:

Youness Limami

Reviewer 2: Anonymous

Reviewer 3:

Byung Joo Kim

Biomolecules 2026, 16(3), 366; https://doi.org/10.3390/biom16030366

Submission received: 1 January 2026 / Revised: 4 February 2026 / Accepted: 6 February 2026 / Published: 28 February 2026

(This article belongs to the Section Natural and Bio-derived Molecules)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma Cells by Harnessing the Expression of microRNAs” investigates the anticancer effects of apigenin in ESCC cell lines (TE-1 and Eca-109), with a particular focus on miRNA-mediated regulatory mechanisms. The study combines functional cellular assays with small RNA sequencing, bioinformatics analyses, and qRT-PCR validation. Overall, the topic is relevant and timely, given the increasing interest in natural compounds and miRNA-based regulatory networks in cancer therapy.

However, several issues need to be addressed before the manuscript can be considered for publication.

Comments:

Several typographical and grammatical errors are present (e.g., “the he human ESCC cell lines”, inconsistent capitalization, spacing issues).
Specify the exact statistical tests used for each experiment in the figure legends.
Some abbreviations are introduced without prior definition (e.g., specific gene symbols, pathway names).
Please ensure all abbreviations are defined at first mention.

While the study identifies multiple differentially expressed miRNAs and associated target genes following apigenin treatment, the conclusions remain largely correlative.

Without direct evidence demonstrating that the observed phenotypic effects (proliferation inhibition, apoptosis, migration) are mediated through specific miRNAs, the mechanistic claims remain speculative. At minimum, the authors should temper their conclusions and clearly acknowledge this limitation in the Discussion.

The rationale for selecting specific miRNAs and target genes for qRT-PCR validation is insufficiently explained.

Please clarify:
Why these particular miRNAs were chosen among the many differentially expressed candidates.
Whether selection was based on fold change, statistical significance, cancer relevance, or prior literature.

The bioinformatics workflow requires further clarification:

The manuscript states that miRNAs with log₂ fold change > 2.0 and p < 0.001 were considered significant; however, volcano plots appear to use different thresholds (e.g., log₂ FC > 1, p < 0.05). Please ensure consistency between the methods section and figure legends.

The wound-healing assay may be confounded by apigenin-induced inhibition of cell proliferation. Since apigenin significantly reduces cell viability, it is difficult to conclude whether reduced wound closure reflects impaired migration or simply decreased proliferation. The authors should clearly acknowledge this limitation in the Results and Discussion sections.

Author Response

Dear Editor

Editorial Office Biomolecules

4 February, 2026

Dear Editor,

Biomolecules-MDPI-(4104293)

“Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of microRNAs.”

We thank you very much for the opportunity to revise the manuscript and appreciate the editors and reviewers for their positive comments. We have read them carefully and revised the manuscript accordingly. Our responses are summarized below.

Reviewer 1:

However, several issues need to be addressed before the manuscript can be considered for publication.

Comments:

Several typographical and grammatical errors are present (e.g., “the he human ESCC cell lines”, inconsistent capitalization, spacing issues).

Specify the exact statistical tests used for each experiment in the figure legends.

Some abbreviations are introduced without prior definition (e.g., specific gene symbols, pathway names).

Please ensure all abbreviations are defined at first mention.

While the study identifies multiple differentially expressed miRNAs and associated target genes following apigenin treatment, the conclusions remain largely correlative.

Without direct evidence demonstrating that the observed phenotypic effects (proliferation inhibition, apoptosis, migration) are mediated through specific miRNAs, the mechanistic claims remain speculative. At minimum, the authors should temper their conclusions and clearly acknowledge this limitation in the Discussion.

The rationale for selecting specific miRNAs and target genes for qRT-PCR validation is insufficiently explained.

Please clarify:

Why these particular miRNAs were chosen among the many differentially expressed candidates.

Whether selection was based on fold change, statistical significance, cancer relevance, or prior literature.

The bioinformatics workflow requires further clarification:

The manuscript states that miRNAs with log₂ fold change > 2.0 and p < 0.001 were considered significant; however, volcano plots appear to use different thresholds (e.g., log₂ FC > 1, p < 0.05). Please ensure consistency between the methods section and figure legends.

Responses to the Reviewer #1:

However, several issues need to be addressed before the manuscript can be considered for publication.

Dear Reviewer, Thank you so much for a detailed evaluation of our manuscript and insightful comments.

The points you’ve raised in your note, we will try our best to address them one by one, with proper logic, evidence, and references.

Several typographical and grammatical errors are present (e.g., “the he human ESCC cell lines”, inconsistent capitalization, spacing issues).

We appreciate the reviewer’s careful attention to detail. The manuscript has been thoroughly revised to correct typographical and grammatical errors, including duplicated words, inconsistent capitalization, and spacing issues. The typographical error you’ve pointed out in your comment has been corrected (“the he human ESCC cell lines”) has been correct in our revised manuscript file Track Changes-biomolecules-4104293 (Line 114). All such errors have been corrected to ensure clarity and consistency throughout the text.

Specify the exact statistical tests used for each experiment in the figure legends.

Thank you for pointing that out. We have revised the figure legends to explicitly include the statistical tests used for each experiment. The relevant statistical tests, such as Student’s t-test and one-way ANOVA with appropriate post hoc analyses, have been specified where applicable in our revised manuscript file, Track Changes-biomolecules-4104293.

Figure 1 (A, B): The statistical test details have been added to the figure legend (Lines 223-225).

Figure 1 (C, D): The statistical details have been added to the figure legend (Lines 228-230).

Figure 2: The statistical details have been incorporated in the revised figure legend (Lines 241-248), and the entire figure legend has been updated.

Figure 3: The statistical details have been added in the figure legend (Lines 269-271).

Figures 4 and 5: As the analysis was conducted using the R package software, the statistical details have already been included in the figure legends (Lines 296, 304) (Lines 329, 335).

Figure 6: The statistical details have been added in the figure legend (Lines 387-389), with the entire figure legend revised.

Figure 7: The statistical details have been included in the figure legend (Lines 420-421), and the entire figure legend has been updated.

Figure 8: The statistical details have been added in the figure legend (Lines 466-467), and the figure legend has been revised in its entirety.

These revisions enhance clarity and improve the transparency and reproducibility of our results.

Some abbreviations are introduced without prior definition (e.g., specific gene symbols, pathway names)

We appreciate the reviewer’s comment and have carefully revised the manuscript to ensure that all abbreviations, including gene symbols and pathway names, are clearly defined at their first occurrence to improve clarity and readability. All has been revised in the Track Changes-biomolecules-4104293 (Line-346-357) and (Line 366-370).

Please ensure all abbreviations are defined at first mention.

Thank you for pointing that out. We have carefully cross-checked our revised manuscript file Track Changes-biomolecules-4104293 to ensure that all abbreviations are clearly defined at their first mention.

While the study identifies multiple differentially expressed miRNAs and associated target genes following apigenin treatment, the conclusions remain largely correlative.

Thank you for this insightful comment. We agree that our study mainly shows correlations between apigenin treatment and changes in miRNA expression and their target genes. While these results point to potential regulatory pathways affected by apigenin in ESCC cells, they do not prove direct causality. We have clarified these limitations in the discussion section and emphasized that future functional studies, such as miRNA overexpression or knockdown experiments, will be needed to confirm the causal roles of specific miRNAs and their targets (Line 706-720) of Track Changes-biomolecules-4104293.

Thank you for pointing out this important suggestion. We agree that, while our study demonstrates that apigenin affects proliferation, apoptosis, and migration in ESCC cells and identifies associated changes in specific miRNAs, we do not provide direct evidence that these phenotypic effects are mediated through those miRNAs. We have revised the discussion section (Line 706-720) of the Track Changes-biomolecules-4104293 to clearly acknowledge this limitation and have tempered our mechanistic claims accordingly. We also highlight that future studies involving targeted manipulation of these miRNAs will be necessary to confirm their causal roles in mediating the effects of apigenin.

Please clarify:

Why these particular miRNAs were chosen among the many differentially expressed candidates.

Dear Reviewer, We selected these miRNAs using predefined and statistically rigorous criteria applied during the bioinformatic analysis. Specifically, miRNAs showing significant differential expression ( , p < 0.05) were identified based on the volcano plots (Figure 5). From this group, miRNAs with the highest fold changes were prioritized for further analysis to focus on those most likely to be biologically relevant. These candidates were subsequently validated by qRT-PCR, and the experimental results closely matched the bioinformatic findings, supporting the reliability of our selection approach. This is discussed in the discussion section (Lines 522-534) Track Changes-biomolecules-4104293.

Whether selection was based on fold change, statistical significance, cancer relevance, or prior literature

Thank you for your comment. The selection was primarily driven by quantitative bioinformatic criteria rather than prior assumptions.

Primary Criteria: fold change and statistical significance

Secondary Criteria: check the consistency with cancer relevance through literature, not the selection key factor

The priority is given to those showing the largest magnitude of change in the volcano plot. This approach ensured an unbiased and data-driven selection of candidate miRNAs. Cancer relevance and existing literature were considered at a later stage to support biological interpretation, but they were not used as initial selection criteria. The selected miRNAs were subsequently validated by qRT-PCR, and their expression patterns were consistent with the bioinformatic analysis

The bioinformatics workflow requires further clarification:

The rationale for selecting specific miRNAs and target genes for qRT-PCR validation is insufficiently explained

Thank you for highlighting this point. We have revised the manuscript to clarify the rationale for selecting specific miRNAs and target genes for qRT-PCR validation. Candidates were chosen based on the magnitude of differential expression observed in the bioinformatic analysis, as shown in the volcano plot, and statistical significance confirmed by qRT-PCR analysis. Their involvement in cancer-related pathways, particularly those associated with proliferation, apoptosis, and metastasis, was also taken into account. Priority was given to miRNAs and target genes with previously reported roles in ESCC or related cancers. This selection strategy has been explicitly detailed in the third paragraph of the Discussion section (Lines 522–534) of Track Changes-biomolecules-4104293.

Thank you for pointing out this discrepancy. We have carefully reviewed the manuscript and figure legends to ensure consistency. The standard practice approach in volcano plot fold change > 1 and p < 0.05. So here in our data, the thresholds used for identifying significantly differentially expressed miRNAs were log₂ fold change > 1.0 and p < 0.05, updated in the Methods section (Line 176), which is inconsistent with corresponding figure legends (Line 329 &335) of Track Changes-biomolecules-4104293.

Thank you for this important observation. We acknowledge that the wound-healing assay may be influenced by the anti-proliferative effects of apigenin, and therefore, reduced wound closure cannot be interpreted solely as a reflection of impaired migration. To address this, we have revised both the Results and Discussion sections to clearly acknowledge this limitation and emphasize that the observed effects likely represent a combination of decreased proliferation and migration in the Results Section (Lines 235-238) and the Discussion Section (Lines 511-513) of the Track Changes-biomolecules-4104293. Future studies using proliferation-independent migration assays, such as live-cell imaging under proliferation-inhibited conditions, will be valuable to dissect the specific contribution of apigenin to cell motility in ESCC cells.

We sincerely thank the reviewer for their insightful suggestions and expert feedback. The manuscript has been thoroughly revised for grammatical checkup and has improved the language overall.

Dear Editor, after addressing all the questions raised by the respected reviewer and improving the linguistics of our manuscript, we sincerely hope that your journal will consider publishing our article in the Journal “Biomolecules” and that our work can contribute to the Natural and Bio-derived Molecules medicinal industry.

Sincerely,

Professor Li Zhiyuan

Principal Investigator (PI)

Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, Center for Cell Lineage Technology and Engineering, Guangzhou Institutes of Biomedicine and Health (GIBH), CAS, Guangzhou, P.R. China.

Email: li_zhiyuan@gibh.ac.cn

Reviewer 2 Report

Comments and Suggestions for Authors

The article: Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of microRNAs

Authors: Nouman Amjad, Majid Muhammad, Sun Zhaojian, Rajesh Basnet, Kashaf Rasool, Wu Linping and Zhiyuan Li

General comments

The study investigates the anticancer effects of apigenin, a natural flavonoid, on esophageal squamous cell carcinoma (ESCC) cell lines (TE-1 and Eca-109). It explores the impact of apigenin on cell proliferation, apoptosis, migration, invasion, and microRNA (miRNA) expression modulation. This study is also notable in that it investigates miRNA-mRNA regulatory networks and the underlying molecular pathways through which apigenin may exert its anticancer effects.

The introduction is well structured, providing well-organized general information that gives a clear representation of the importance of the topic addressed.

The techniques used are well described in the materials and methods section. The methods addressed are clear and representative of the objective set. The results are generally presented appropriately, with many details that help to understand the molecular processes analyzed.

The discussions and conclusions are relevant and well structured. The directions and perspectives indicate that further studies are needed to validate the direct miRNA-mRNA interactions observed and to explore the clinical applications of apigenin in combination with existing conventional treatments. Apigenin shows promise as a therapeutic or adjuvant agent for ESCC due to its low toxicity and ability to enhance the efficacy of conventional treatments while reducing their side effects.

Recommendations

I believe that the results presented in the graphs could be rearranged slightly. My observations suggest an improvement in the presentation in Figure 4B, where there are overlaps and lines.

Also, in the same context, the names of the cell types on which the effects are analyzed should be included in the titles of the graphs for both Figures 6 and 7.

I consider the paper suitable for publication in the Biomolecules Journal.

Author Response

Dear Editor

Editorial Office Biomolecules

4 February, 2026

Dear Editor,

Biomolecules-MDPI-(4104293)

“Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of microRNAs.”

Reviewer 2:

The article: Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of microRNAs

Authors: Nouman Amjad, Majid Muhammad, Sun Zhaojian, Rajesh Basnet, Kashaf Rasool, Wu Linping and Zhiyuan Li

General comments

The introduction is well structured, providing well-organized general information that gives a clear representation of the importance of the topic addressed.

Recommendations

I believe that the results presented in the graphs could be rearranged slightly. My observations suggest an improvement in the presentation in Figure 4B, where there are overlaps and lines.

Also, in the same context, the names of the cell types on which the effects are analyzed should be included in the titles of the graphs for both Figures 6 and 7.

I consider the paper suitable for publication in the Biomolecules Journal.

Response to Reviewer

The introduction is well structured, providing well-organized general information that gives a clear representation of the importance of the topic addressed.

Dear Reviewer, Thank you for your positive and constructive evaluation of our manuscript. We appreciate the acknowledgment of the study design, methodological clarity, and the relevance of our work on miRNA–mRNA regulatory networks in the context of apigenin’s anticancer effects in ESCC. We have also discussed these limitations in the discussion section Track Changes-biomolecules-4104293 (Lines 706-720).

Recommendations

I believe that the results presented in the graphs could be rearranged slightly. My observations suggest an improvement in the presentation in Figure 4B, where there are overlaps and lines.

Thank you, Sir, for pointing this out. We have carefully reviewed Figure 4B and checked its clarity. The figure is already provided in SVG format, which preserves resolution and prevents line overlap upon zooming. We have updated the figure in our revised manuscript, i.e., Track Changes-biomolecules-4104293 (Line 295-26). The figure has been prepared in accordance with the journal’s guidelines. We will further consult with the Associate Editor regarding further improvements to enhance its clarity.

Also, in the same context, the names of the cell types on which the effects are analyzed should be included in the titles of the graphs for both Figures 6 and 7.

Thanks for this valuable suggestion. We have revised Figures 6, 7, and 8 to include the names of the corresponding cell lines in the titles of the graphs in our revised version, Track Changes-biomolecules-4104293 (Line 379, 414, 460). Which improves clarity and ensures that the experimental context is immediately clear to the reader.

I consider the paper suitable for publication in the Biomolecules Journal.

We sincerely thank the reviewer for the positive evaluation and for considering our manuscript suitable for publication in Biomolecules. We truly appreciate the time and effort spent reviewing our work and the thoughtful feedback provided, which has helped us improve the manuscript.

Sincerely,

Professor Li Zhiyuan

Principal Investigator (PI)

Email: li_zhiyuan@gibh.ac.cn

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the anticancer effects of apigenin, a naturally occurring flavonoid with low toxicity, in esophageal squamous cell carcinoma (ESCC) cell lines (TE-1 and Eca-109). The authors demonstrate that apigenin suppresses proliferation, clonogenicity, migration, and invasion while promoting apoptosis. Using small RNA sequencing, the study further profiles apigenin-induced alterations in miRNA expression and predicts downstream target genes, which are analyzed through GO and KEGG pathway enrichment and partially validated by qRT-PCR. The authors propose that apigenin exerts its antitumor effects through coordinated miRNA–mRNA regulatory networks.

Overall, the topic is of interest, particularly given the growing attention to traditional medicine–derived compounds and miRNA-mediated cancer regulation. However, several critical mechanistic and interpretational issues limit the strength and translational impact of the current study.

1.While the manuscript identifies multiple apigenin-regulated miRNAs and correlates these changes with altered expression of cancer-related genes, causal evidence linking specific miRNAs to the observed antitumor phenotypes is insufficient. The study relies primarily on expression profiling and bioinformatic predictions without functional validation. To support the central claim that miRNA modulation mediates apigenin’s anticancer effects, the authors should perform gain- or loss-of-function experiments (e.g., miRNA mimics, inhibitors, or rescue assays) to demonstrate that manipulation of key miRNAs alters the cellular response to apigenin.

2.In several cases, the manuscript reports that certain miRNAs and their predicted target genes are regulated in the same direction following apigenin treatment. This observation is inconsistent with the canonical miRNA-mediated repression model. The authors should provide a clear mechanistic explanation for this phenomenon (e.g., indirect regulation, feedback loops, or non-canonical miRNA functions) or supply additional experimental evidence to substantiate these regulatory relationships. Without clarification, the interpretation of the miRNA–mRNA network remains ambiguous.

3.The miRNA expression profiles altered by apigenin differ substantially between TE-1 and Eca-109 cells. However, the biological basis and implications of this cell line–specific response are not adequately discussed. The authors should clarify whether these differences reflect intrinsic molecular heterogeneity of ESCC (such as differentiation status, genetic background, or baseline miRNA landscapes) and discuss how this variability affects the generalizability of the proposed mechanism.

4.Although apigenin is widely present in multiple traditional medicinal herbs, the manuscript does not sufficiently contextualize its findings within the framework of traditional medicine–based cancer therapy. The authors should clearly identify relevant traditional medicinal sources or systems in which apigenin is found and expand the Discussion to explain why mechanistic studies of traditional medicine–derived compounds are important. In particular, the potential role of miRNA-mediated regulation as a molecular basis for the multi-target effects of traditional medicines should be more explicitly addressed.

5.The Discussion section does not adequately acknowledge the limitations of the study. In particular, the authors should explicitly discuss that: the findings are primarily based on in vitro cell line models, most miRNA–target interactions are inferred from bioinformatic predictions rather than direct functional validation, and the lack of in vivo or clinical data limits the translational relevance of the conclusions. A dedicated paragraph outlining these limitations and proposing future research directions (e.g., in vivo validation or clinical correlation) is strongly recommended.

Author Response

Dear Editor

Editorial Office Biomolecules

4 February, 2026

Dear Editor,

Biomolecules-MDPI-(4104293)

“Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of microRNAs.”

Reviewer 3:

Response to Reviewer

Dear Reviewer, Thank you so much for a detailed evaluation of our manuscript and insightful comments.

The points you’ve raised in your note, we will try our best to address them one by one, with proper logic, evidence, and references.

Thank you, dear Reviewer, for your constructive comment. We agree that the current study does not provide direct functional evidence demonstrating that specific miRNAs causally mediate the antitumor effects of apigenin. Our conclusions are based on integrated miRNA expression profiling, bioinformatic prediction of miRNA–mRNA interactions, pathway enrichment analyses, and targeted qRT-PCR validation, which together identify candidate regulatory networks associated with apigenin treatment rather than definitive causal relationships.

We have therefore revised the manuscript to temper our central claims and to clearly state that the identified miRNAs represent potential mediators of apigenin’s anticancer effects. The study is now positioned as a comprehensive, hypothesis-generating analysis that defines biologically relevant miRNA–mRNA signatures and signaling pathways responsive to apigenin in ESCC cells. We also explicitly acknowledged the limitations in the Discussion section of our revised manuscript Track Changes-biomolecules-4104293 (Lines 707-721) that functional validation using gain- or loss-of-function approaches, such as miRNA mimics, inhibitors, or rescue assays, will be required in future studies to establish direct causality and to delineate the precise contribution of individual miRNAs to the observed antitumor phenotypes.

Importantly, by combining phenotypic assays with transcriptomic profiling, pathway analysis, and experimental validation of selected miRNAs and target genes, our study provides a robust framework for prioritizing key miRNA–mRNA axes for subsequent mechanistic investigation.

In classical canonical miRNA regulation, mature miRNAs guide Argonaute-containing silencing complexes to complementary sites within the 3′ untranslated region of target mRNAs, resulting in translational repression and/or mRNA destabilization. Consistent with this paradigm, our data demonstrate a similar pattern: as miRNA expression is upregulated, the corresponding potential target mRNAs are downregulated, and vice versa.

For clarity and review, we have provided screenshots of our Excel data, which corroborate the results obtained from both bioinformatic analyses and qRT-PCR validation.

TE-1-Upregulated & downregulated miRNAs-mRNAs (Canonical Pattern)

Added in the attached PDF file

Eca-109-Upregulated & downregulated miRNAs-mRNAs (Canonical Pattern)

Added in the attached PDF file

While we also observed some non-canonical patterns, those were not included in the presented dataset to maintain consistency and emphasize the classical inverse relationship characteristic of canonical miRNA-mediated regulation. This observation has been further discussed in the Discussion section of Track Changes-biomolecules-4104293 (Lines 681–705).

We appreciate the reviewer’s insightful comment regarding the cell line–specific differences in miRNA expression profiles following apigenin treatment. Indeed, TE-1 and Eca-109 cells exhibit distinct molecular backgrounds, including differences in differentiation status, genetic alterations, and baseline miRNA expression landscapes, which likely contribute to the observed variability in response. Such intrinsic heterogeneity is characteristic of esophageal squamous cell carcinoma (ESCC) and underscores the context-dependent nature of miRNA-mediated regulation. While our study highlights common canonical miRNA–mRNA interactions that are consistent across both cell lines, we acknowledge that the magnitude and specific patterns of miRNA modulation may differ due to these intrinsic factors. We have now addressed this point in the Discussion section of the Track Changes-biomolecules-4104293 (Lines 664–678), emphasizing that while the proposed mechanism is broadly relevant, cell line–specific differences should be considered when extrapolating to other ESCC contexts or patient samples.

We appreciate the reviewer’s comment. Apigenin is a flavonoid found in several traditional medicinal herbs, which have been used for their anti-inflammatory and anticancer properties. Studying the molecular mechanisms of such traditional medicine–derived compounds is essential to provide a scientific basis for their therapeutic effects. Our findings indicate that miRNA-mediated regulation may underlie the multi-target actions of apigenin, offering insight into how traditional compounds can modulate multiple oncogenic and tumor-suppressive pathways in ESCC. We have updated the Discussion section of the Track Changes-biomolecules-4104293 (Lines 492–504) to highlight how miRNA networks may mediate the multi-target effects of traditional medicine–derived compounds and how understanding these mechanisms can guide therapeutic strategies in ESCC.

We thank the reviewer for this important comment. As illustrated in response to your first comment, we acknowledge several limitations in our study. First, the findings are based primarily on in vitro ESCC cell line models, which may not fully capture the complexity of tumor biology in patients. Second, most miRNA–target interactions were inferred from bioinformatic predictions; although qRT-PCR validated expression trends, direct functional assays were not performed to confirm causality. Third, the absence of in vivo or clinical data limits the immediate translational relevance of our conclusions. Future studies should incorporate in vivo models, patient-derived samples, and functional validation of key miRNA–mRNA interactions to strengthen mechanistic insights and therapeutic applicability. These limitations are discussed in the Discussion section of the Track Changes-biomolecules-4104293 (Lines 707-721).

We sincerely thank the reviewer for their valuable suggestions and constructive comments. We have thoroughly revised our article for grammatical checkup and improved the language overall.

Dear Editor, after addressing all the questions raised by the respected reviewers and improving the linguistics of our manuscript, we sincerely hope that your journal will consider publishing our article in the Journal “Biomolecules” and that our work can contribute to the Natural and Bio-derived Molecules medicinal industry.

Sincerely,

Professor Li Zhiyuan

Principal Investigator (PI)

Email: li_zhiyuan@gibh.ac.cn

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I went through the revised version of the manuscript and noticed that the authors have addressed all the issues raised and significantly improved the manuscript. Therefore, my recommendation would be to accept the manuscript in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

It is well revised.

Article Menu

Apigenin Inhibits the Growth of Esophageal Squamous Cell Carcinoma (ESCC) Cells by Harnessing the Expression of MicroRNAs

Further Information

Guidelines

MDPI Initiatives

Follow MDPI