Review Reports - In Vitro Effects of Mir-6757-3p Modulation and AT9283 Treatment on AURKA/AURKB Expression and Apoptosis-Related Responses in A549 and BEAS-2B Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the Authors,

We thank the authors for submitting their manuscript to *Biomedicines*. The study addresses an interesting topic and presents potentially relevant *in vitro* observations regarding miR-6757 and responses involving Aurora kinase. A positive aspect of the experimental design is the inclusion of both A549 lung cancer cells and BEAS-2B cells as a non-tumorigenic comparison model, allowing the responses observed in the cancer model to be analyzed in parallel with a non-tumorigenic cellular model.

The integration of gene expression analyses, protein levels, and cellular assays also provides complementary information on the effects under investigation. The inclusion of BEAS-2B cells is relevant and could potentially be better reflected in the title. However, several points regarding the consistency between the experimental design, the Materials and Methods section, the Results, and the data interpretation need to be clarified before the manuscript can be considered for publication.

1. Lines 2-5: Regarding the title, the authors might consider revising certain terms to more accurately reflect the experimental design and the scope of the findings. The term "co-treatment" could be reconsidered if miR-6757 modulation and Aurora kinase inhibition were evaluated separately. Furthermore, expressions such as "AURKA/B signaling pathway" and "modulates apoptosis" might imply mechanistic or functional effects beyond those directly assessed; more descriptive terms, such as "AURKA/AURKB expression" and "apoptosis-related responses," could be considered.

For instance, the authors might consider a title such as: "In vitro effects of miR-6757 modulation and Aurora kinase inhibition on AURKA/AURKB expression and apoptosis-related responses in A549 cells, using BEAS-2B cells as a non-tumor control." This wording may more accurately represent the experimental approach, avoiding mechanistic implications that might go beyond the presented data.

2. Line 87: The citation appears duplicated and in different formats: "programmed cell death (17-20, 24) [17-21]." Similar citation and formatting inconsistencies are present in other sections of the manuscript. The authors should systematically review all in-text citations to ensure they comply with the journal's style.

3. Lines 108-110: The description should be revised to clearly distinguish between gene expression analysis via qRT-PCR and protein measurements via ELISA.

4. Line 119: The Aurora kinase inhibitor is identified as AT9383, whereas AT9283 is used elsewhere in the manuscript. The authors should verify the correct compound designation and use it consistently throughout the manuscript.

5. Lines 143-157 and Table 1: Gene and protein nomenclature is inconsistent throughout the manuscript, featuring varied formats such as BAX/Bax, BCL2/Bcl-2, CASP3/caspase-3, and MTOR/mTOR. The authors should revise and standardize the nomenclature throughout the manuscript.

6. Lines 153-154 and 177-184: The qRT-PCR methodology states that reactions were performed "in triplicate," whereas the Statistical Analysis section refers to biological triplicates and independent experiments. The authors should clarify whether the qRT-PCR measurements involved technical replicates, biological replicates, or both, and report this consistently throughout the manuscript.

7. Lines 160-162: An ELISA assay is described for MDA, BAX, BCL-2, CASPASE-3, and TAS. However, Figures 3 and 4 present ELISA measurements for AURKA, AURKB, BAX, BCL2, and CASP3, while MDA and TAS do not appear in the reported results. The authors should revise the Methods and Results sections to ensure that all ELISA measurements are described and reported consistently. If MDA and TAS were measured as part of this study, the authors should clarify whether these data are included in the manuscript.

8. Lines 252-258: The wound healing assay results are interpreted using terms such as "cellular damage," "protective effects," and "cellular stress." Such interpretations would generally require additional experimental evidence, as they are not directly assessed by the wound healing assay used in this study. The authors might consider describing these findings primarily in terms of wound closure and cell migration. Similar interpretations appear in other sections of the manuscript, including the Abstract and the Discussion, and should be revised accordingly.

Author Response

Comment 1

Lines 2–5: Regarding the title, the authors might consider revising certain terms to more accurately reflect the experimental design and the scope of the findings. The term "co-treatment" could be reconsidered if miR-6757 modulation and Aurora kinase inhibition were evaluated separately. Furthermore, expressions such as "AURKA/B signaling pathway" and "modulates apoptosis" might imply mechanistic or functional effects beyond those directly assessed; more descriptive terms, such as "AURKA/AURKB expression" and "apoptosis-related responses," could be considered.

Response

We thank the reviewer for this important suggestion. We agree that the previous title could imply a combined treatment design and mechanistic effects that were not directly established in the present study. Accordingly, the title has been revised to more accurately reflect the experimental design and the scope of the findings.

The revised title is:

“In vitro effects of miR-6757 modulation and AT9283 treatment on AURKA/AURKB expression and apoptosis-related responses in A549 and BEAS-2B cells.”

The terms “co-treatment,” “AURKA/B signaling pathway,” and “modulates apoptosis” were removed to avoid implying direct mechanistic or functional relationships beyond the experimental evidence. The inclusion of BEAS-2B cells as a non-tumorigenic comparison model is also now explicitly reflected in the title.

Comment 2

Line 87: The citation appears duplicated and in different formats: "programmed cell death (17-20, 24) [17-21]." Similar citation and formatting inconsistencies are present in other sections of the manuscript. The authors should systematically review all in-text citations to ensure they comply with the journal's style.

Response

We thank the reviewer for identifying this citation-formatting problem. The manuscript was systematically reviewed for duplicated, inconsistent, and incorrectly formatted in-text citations. The duplicated citation and inconsistent citation formats were corrected, and the references were revised to follow a consistent citation style throughout the manuscript.

Comment 3

Lines 108–110: The description should be revised to clearly distinguish between gene expression analysis via qRT-PCR and protein measurements via ELISA.

Response

We agree with the reviewer. The Materials and Methods section was revised to clearly distinguish between transcriptional and protein-level analyses.

Specifically, the qRT-PCR section now describes the analysis of BAX, BCL2, CASP3, CASP9, MTOR, AURKA, and AURKB mRNA expression, whereas the ELISA section separately describes the measurement of AURKA, AURKB, BAX, BCL-2, and CASPASE-3 protein levels.

In addition, the Results section was revised so that qRT-PCR findings are presented as gene-expression results and ELISA findings are explicitly described as protein-level measurements. This distinction is now maintained throughout the manuscript.

Comment 4

Line 119: The Aurora kinase inhibitor is identified as AT9383, whereas AT9283 is used elsewhere in the manuscript. The authors should verify the correct compound designation and use it consistently throughout the manuscript.

Response

We thank the reviewer for pointing out this inconsistency. The correct compound used in the present study is AT9283 (Selleckchem, S1134, Houston, USA).

The incorrect designation “AT9383” was corrected to AT9283 throughout the manuscript. The compound is now consistently referred to as AT9283 in the title, Materials and Methods, Results, Discussion, figure legends, and other relevant sections.

Comment 5

Lines 143–157 and Table 1: Gene and protein nomenclature is inconsistent throughout the manuscript, featuring varied formats such as BAX/Bax, BCL2/Bcl-2, CASP3/caspase-3, and MTOR/mTOR. The authors should revise and standardize the nomenclature throughout the manuscript.

Response

We agree with the reviewer. The manuscript was carefully reviewed to standardize gene and protein nomenclature throughout the text, tables, figures, and figure legends.

Gene symbols are now consistently presented using the standardized gene nomenclature, including BAX, BCL2, CASP3, CASP9, MTOR, AURKA, and AURKB, whereas protein-level measurements are explicitly described as protein levels of the corresponding targets.

This revision was also applied to the Results, Discussion, Conclusion, and figure legends to improve consistency and avoid ambiguity between gene-expression and protein-level findings.

Comment 6

Lines 153–154 and 177–184: The qRT-PCR methodology states that reactions were performed "in triplicate," whereas the Statistical Analysis section refers to biological triplicates and independent experiments. The authors should clarify whether the qRT-PCR measurements involved technical replicates, biological replicates, or both, and report this consistently throughout the manuscript.

Response

We thank the reviewer for requesting clarification. In the revised manuscript, we have removed the ambiguous wording “performed in triplicate” and explicitly stated that the qRT-PCR analyses were performed using three independent biological replicates (n = 3).

The Statistical Analysis section has also been revised to consistently indicate that the experiments were performed using three independent biological replicates, with data presented as mean ± standard deviation.

Accordingly, the qRT-PCR Methods, Statistical Analysis, and relevant figure legends now consistently report n = 3 independent biological replicates.

Comment 7

Lines 160–162: An ELISA assay is described for MDA, BAX, BCL-2, CASPASE-3, and TAS. However, Figures 3 and 4 present ELISA measurements for AURKA, AURKB, BAX, BCL2, and CASP3, while MDA and TAS do not appear in the reported results. The authors should revise the Methods and Results sections to ensure that all ELISA measurements are described and reported consistently. If MDA and TAS were measured as part of this study, the authors should clarify whether these data are included in the manuscript.

Response

We thank the reviewer for identifying this inconsistency. We have carefully revised the ELISA Methods and Results sections to ensure that only the protein measurements actually reported in the manuscript are described.

The references to MDA and TAS were removed from the ELISA Methods section, as these measurements are not included in the current Results or figures. The revised ELISA section now describes the measurement of AURKA, AURKB, BAX, BCL-2, and CASPASE-3 protein levels, which correspond to the data presented in Figures 3 and 4.

Thus, the revised manuscript now provides consistency between the ELISA methodology and the reported experimental results.

Comment 8

Lines 252–258: The wound healing assay results are interpreted using terms such as "cellular damage," "protective effects," and "cellular stress." Such interpretations would generally require additional experimental evidence, as they are not directly assessed by the wound healing assay used in this study. The authors might consider describing these findings primarily in terms of wound closure and cell migration. Similar interpretations appear in other sections of the manuscript, including the Abstract and the Discussion, and should be revised accordingly.

Response

We fully agree with the reviewer. We acknowledge that a scratch/wound-healing assay alone does not allow direct conclusions regarding cellular damage, protective effects, cellular stress, or apoptosis.

Accordingly, the interpretation of the wound-healing findings was revised throughout the manuscript. In the Results section, the findings are now described primarily in terms of wound closure and time-dependent changes in the wound-healing response.

In the Discussion, statements referring to “cellular damage,” “protective effects,” and “cellular stress” in relation to the scratch assay were removed or replaced with more descriptive and appropriately cautious terminology. The revised text explicitly states that the scratch assay does not allow direct conclusions regarding cellular damage, protection, or apoptosis.

Furthermore, the Abstract and Conclusion were revised to avoid overinterpretation of the wound-healing findings. The revised manuscript now states that the observed treatments were associated with differences in wound closure and that these findings should be interpreted within the limitations of the assay.

We also clarified that the molecular changes observed following AT9283 treatment should not be interpreted as evidence of a specific Aurora kinase-mediated apoptotic mechanism because AT9283 is a multi-target kinase inhibitor and no direct apoptosis assay was performed.

We believe these revisions have substantially improved the consistency between the experimental design and the interpretation of the wound-healing results.

Reviewer 2 Report

Comments and Suggestions for Authors

The comments and suggestions for authors are presented in REVIEW REPORT 4534985 V1.PDF

Comments for author File: Comments.pdf

Author Response

Major Comments

Reviewer Comment 1

The central contradiction between the apoptosis/migration data and the conclusion that miR-6757 acts as a tumor suppressor must be resolved. The mimic appears to promote survival, be anti-apoptotic and stimulate migration, whereas the inhibitor shows the opposite effects. The conclusion should either be reinterpreted or supported with further experiments.

Response: We agree with the reviewer. The previous wording suggesting that miR-6757 has a tumor-suppressive role was too strong and did not adequately reflect the direction of the molecular and wound-closure findings. We therefore removed the tumor-suppressive conclusion. The revised manuscript now states that the present findings do not allow miR-6757 to be classified as either tumor-suppressive or oncogenic and that the observed effects may be context-dependent. We also avoided interpreting wound closure as direct evidence of apoptosis, protection, or cellular damage.

Revision made: Discussion and Conclusion were revised to use association-based and context-dependent language. The revised Conclusion explicitly states that the study does not establish a causal mechanism or direct molecular targeting.

Reviewer Comment 2

The suggested mechanism 'through AURKA/B signaling' is internally inconsistent. The inhibitor increases AURKA/AURKB expression but is also associated with apoptosis/cellular damage. The text should therefore use association rather than causation unless causal experiments are performed.

Response: We agree. The revised manuscript no longer presents AURKA/AURKB as an established causal pathway mediating the observed responses. AT9283 is described as a multi-target kinase inhibitor and is treated as a pharmacological comparator rather than as a selective mechanistic probe. The interpretation of AT9283-associated molecular changes has also been explicitly limited because no direct apoptosis assay was performed.

Revision made: The title, Abstract, Results, Discussion, and Conclusion were revised to emphasize an association between miR-6757 modulation, Aurora kinase expression, and apoptosis-related molecular responses. Statements implying a specific Aurora kinase-mediated apoptotic mechanism were removed or softened.

Reviewer Comment 3

There is no evidence that AURKA or AURKB are direct targets of miR-6757. At minimum, bioinformatic target prediction should be provided and ideally a 3′UTR luciferase reporter assay should be included.

Response: We agree that the current experiments do not establish direct targeting. We did not perform a luciferase reporter assay or a dedicated direct-target validation experiment, and we therefore do not claim that AURKA or AURKB are direct targets of miR-6757. Instead, the revised manuscript explicitly identifies this as a limitation and states that bioinformatic prediction and luciferase reporter assays would be required to determine whether the observed relationship involves direct post-transcriptional regulation.

Revision made: Direct-target language was removed from the interpretation. The Discussion now explicitly states that the present findings do not establish whether AURKA or AURKB are direct targets and identifies target-prediction and luciferase assays as future work.

Reviewer Comment 4

The title refers to a 'co-treatment' with miR-6757 and an Aurora kinase inhibitor, but no combination group was actually tested. Either perform a combination experiment or remove 'co-treatment' from the title and abstract.

Response: We agree. No group received miR-6757 mimic/inhibitor together with AT9283. Therefore, the term 'co-treatment' was inaccurate and has been removed. The revised manuscript describes the interventions as miR-6757 mimic, miR-6757 inhibitor, and AT9283 treatment, without implying a combination arm.

Revision made: The title was changed to 'In vitro effects of miR-6757 modulation and AT9283 treatment on AURKA/AURKB expression and apoptosis-related responses in A549 and BEAS-2B cells.' Corresponding wording in the Abstract and throughout the manuscript was also corrected.

Reviewer Comment 5

Which arm of miR-6757 was investigated? The Introduction mentions both miR-6757-3p and miR-6757-5p, whereas the Methods refer to miR-6757-3p.

Response: We agree that this distinction must be explicit. The present study specifically evaluated the miR-6757-3p arm. The miR-6757-5p literature is mentioned only to provide context and is not presented as the experimentally investigated arm.

Revision made: The Methods now explicitly state: 'The present study specifically evaluated the miR-6757-3p arm.' The revised manuscript also distinguishes the published biological contexts of miR-6757-3p and miR-6757-5p.

Reviewer Comment 6

There is no direct apoptosis assay and no viability or proliferation assay. Marker expression alone does not prove apoptosis, and wound closure can be affected by proliferation. A direct apoptosis assay and viability/proliferation assay should be added or the claims substantially reduced.

Response: We agree with the reviewer. Additional functional apoptosis and viability/proliferation assays were not performed within the scope of the study. Accordingly, we have substantially reduced the claims. The revised Results explicitly state that changes in apoptosis-related molecular markers do not by themselves establish the occurrence of apoptosis. The scratch assay is now described as a wound-closure/wound-healing response rather than as direct evidence of migration, cellular damage, protection, or apoptosis.

Revision made: The Discussion now acknowledges the absence of Annexin V/PI or other direct apoptosis assays and the absence of a dedicated viability/proliferation assay. These limitations are incorporated into the interpretation and Conclusion.

Reviewer Comment 7

Transfection efficiency and knockdown/overexpression were not verified. The inhibitor does not reduce miR-6757 in Figure 2A, while AT9283 appears to increase miR-6757 by 6–7-fold. The transfection protocol and efficiency should be clarified and the unexpected AT9283 effect explained.

Response: We acknowledge this limitation. The revised manuscript does not interpret the observed miR-6757 expression changes as definitive proof of functional knockdown or overexpression. We explicitly state that a dedicated functional transfection-efficiency assay was not independently performed. The unexpected direction of miR-6757 expression following some treatments, including AT9283, is therefore interpreted cautiously and no specific mechanistic explanation is assigned to the AT9283-associated increase.

Revision made: The Discussion was revised to state that the miR-6757 expression profile did not uniformly correspond to the expected direction of modulation and that the findings should not be considered definitive evidence of successful functional knockdown or overexpression. The Methods now provides the inhibitor/mimic identifiers and specifies the miR-6757-3p assay.

Reviewer Comment 8

There is a mismatch between the Methods and Results regarding ELISA and oxidative-stress data. MDA/TAS are listed in the reviewer report/previous version, while AURKA/AURKB are not consistently described. Figure 5 also appears to have been interpreted as cellular damage although it is a wound-healing assay.

Response: We agree and have aligned the endpoint descriptions. The revised ELISA Methods explicitly state that AURKA, AURKB, BAX, BCL-2, and CASPASE-3 protein levels were measured. MDA and TAS are no longer presented as study endpoints. Figure 5 is now described as a time-dependent wound-closure assay, and the Discussion explicitly states that slower or faster wound closure cannot by itself be interpreted as cellular damage, protection, or apoptosis.

Revision made: The Methods, Results, Figure 5 legend, and Discussion were revised to ensure that the described endpoints correspond to the actual analyses.

Reviewer Comment 9

The protein-level concordance is overstated, and the qRT-PCR reporting/statistics should be improved according to MIQE. Only GAPDH is used as a reference gene; amplification efficiencies and Ct/ΔCt information are not reported; n=3 is small; normality/homogeneity tests and exact statistical tests/post-hoc procedures should be specified.

Response: We agree that the wording concerning protein-level concordance needed to be more cautious. The revised Results now state that the ELISA findings were only partially concordant and that not all comparisons reached statistical significance. Regarding qRT-PCR reporting, the revised Methods explicitly identify GAPDH as the reference gene for mRNA and RNU6B_13 as the endogenous control for miR-6757-3p, describe the 2−ΔCt/2−ΔΔCt calculations, and specify three independent biological replicates (n=3). We also explicitly disclose that amplification efficiencies were not independently determined, which is now acknowledged as a methodological limitation. The statistical section specifies GraphPad Prism 8, one-way or two-way ANOVA as appropriate, post-hoc testing, mean ± SD, and n=3. We have not claimed that additional normality/homogeneity testing was performed where it was not documented.

Revision made: The revised Methods and figure legends provide clearer information on n, normalization, calculation of relative expression, statistical procedures, and variability. The Discussion also acknowledges the limitation associated with the small number of biological replicates and the absence of independently determined amplification efficiencies.

Minor Comments

Reviewer Comment 10

AT9283 is a multi-targeted inhibitor, not a selective Aurora kinase inhibitor; it also acts on JAK2/3 and ABL. In addition, 'AT9383' appears in the Materials section and should be corrected to AT9283.

Response: We agree. The compound is now consistently identified as AT9283 and is described as a multi-target kinase inhibitor with activity against Aurora A and Aurora B. The manuscript no longer refers to it as a selective Aurora kinase inhibitor.

Revision made: The Materials and Methods section and the relevant Results, Discussion, title, and figure legends were checked for consistent use of 'AT9283.'

Reviewer Comment 11

The legend for Figure 5 does not correspond to the figure and uses mean ± SEM whereas the manuscript uses mean ± SD. The legend should accurately describe the panels and the AT9283 group.

Response: We agree. The Figure 5 legend was rewritten to describe the actual wound-closure images and quantitative assessment and to use mean ± SD consistently with the manuscript. The AT9283 treatment group is also explicitly identified.

Revision made: The revised Figure 5 legend states that wound closure was assessed at 0, 4, 8, 24, and 48 h and that data are presented as mean ± SD.

Reviewer Comment 12

The phrase 'network-prioritized candidate genes' is unsupported because no network or bioinformatic prioritization analysis is described.

Response: We agree. Because no network-based prioritization analysis was performed, this terminology was removed to avoid implying an analysis that was not conducted.

Revision made: The unsupported 'network-prioritized candidate genes' wording was removed.

Reviewer Comment 13

Include statements concerning STR authentication and mycoplasma testing for A549 and BEAS-2B.

Response: We agree. Independent STR-based authentication and mycoplasma testing were not performed in the present study. Rather than implying that these procedures were performed, we now state this transparently as a methodological limitation.

Revision made: The Cell Culture section now states that STR profiling and mycoplasma testing were not independently performed. This limitation is also mentioned in the Discussion.

Reviewer Comment 14

Because pro-apoptotic molecular effects were also observed in the normal/non-tumorigenic BEAS-2B cell line, address what this means for a therapeutic window and whether the effects are specific to A549.

Response: We agree. The revised Discussion now explicitly states that similar molecular responses in BEAS-2B do not establish a cancer-cell-specific effect or a therapeutic window. The findings are therefore interpreted as cell-context-dependent rather than as evidence of selective activity against A549 cells.

Revision made: The Discussion was revised to distinguish molecular responses observed in A549 from those in BEAS-2B and to avoid implying cancer-cell selectivity or therapeutic benefit.

Reviewer Comment 15

The references should be checked and corrected. Some citations appear irrelevant or mismatched, including references 40–41, and there are duplicated/mixed citation-numbering problems.

Response: We agree. The reference list and in-text citations were reviewed. The previously used references 40–41 were not retained as support for the apoptosis-related endpoint interpretation. Citation numbering and matching were revised so that statements are supported by the cited literature. The reference list was also checked for duplicated or mismatched citations.

Revision made: References and citation placement were revised throughout the manuscript, particularly in the Introduction and Discussion.

Reviewer Comment 16

There may be an inappropriate self-citation. Reference 23 is only weakly related to the present topic; either explain its relevance or remove it. Reference 24 is more directly relevant.

Response: We agree. The relevance of the authors' previous work was reconsidered, and weakly related self-citation was not retained merely for citation purposes. The manuscript now relies on references that directly support the statements being made.

Revision made: The relevant citation was reconsidered and the Discussion/Introduction wording was adjusted to maintain appropriate citation relevance.

Reviewer Comment 17

Provide a data availability statement and provide raw qRT-PCR Ct values and, if possible, a MIQE checklist as supplementary material.

Response: We agree with the request for a clear data availability statement. A Data Availability Statement has now been added. The manuscript states that the datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. The revised Methods also provide additional qRT-PCR reporting details and explicitly acknowledge that amplification efficiencies were not independently determined. We have not represented a MIQE checklist or raw Ct dataset as supplementary material because these materials were not added to the revised manuscript.

Revision made: A Data Availability Statement was added: 'The datasets generated and/or analysed during the current study are available from Hasan Dağlı, the corresponding author, on reasonable request.'

Reviewer Recommendations

Recommendation 1

Address the central contradiction and reconcile the apoptosis, migration, and time-course data with the biological interpretation.

Response: Implemented. The tumor-suppressive interpretation was removed, and the findings are now presented as associations with context-dependent responses. Wound closure is not interpreted as direct evidence of apoptosis or cellular damage.

Recommendation 2

Include target validation and, if possible, a 3′UTR luciferase assay for AURKA/AURKB; clarify the −3p and −5p arms.

Response: The manuscript now clearly specifies miR-6757-3p and explicitly acknowledges that direct target validation was not performed. Luciferase and bioinformatic validation are identified as future studies.

Recommendation 3

Introduce direct functional endpoints, including apoptosis and viability/proliferation assays, and verify transfection and knockdown/overexpression.

Response: These additional experiments were not performed in the present revision. Instead, the claims were substantially reduced, the absence of direct functional assays was stated as a limitation, and the lack of an independent transfection-efficiency assay was disclosed.

Recommendation 4

Correct the title and abstract, remove 'Co-Treatment,' replace causal 'via AURKA/B signaling' language with association-based wording, and ensure protein concordance is accurately reported.

Response: Implemented. The title was revised, causal language was reduced, and protein-level findings are now described as partially concordant rather than uniformly confirmatory.

Recommendation 5

Make the Methods and Results consistent regarding ELISA/MDA/TAS and clarify Figure 5.

Response: Implemented. The revised ELISA endpoint list includes AURKA, AURKB, BAX, BCL-2, and CASPASE-3; MDA/TAS are not presented as endpoints; and Figure 5 is described as a wound-closure assay.

Recommendation 6

Improve statistics and qPCR reporting, correct AT9283 terminology, clean the reference list, and include authentication/mycoplasma and data-availability statements.

Response: Implemented to the extent supported by the available data. AT9283 terminology, reference/citation consistency, STR/mycoplasma disclosure, data availability, biological replicate reporting, normalization, and amplification-efficiency limitation were addressed. We have not claimed that undocumented normality/homogeneity tests or a MIQE checklist were completed.

Overall Revision Summary

Removed the unsupported tumor-suppressive classification of miR-6757 and adopted a context-dependent interpretation.

Removed causal/mechanistic claims implying that the observed effects occur specifically 'through AURKA/AURKB signaling.'

Removed 'co-treatment' terminology because no combination treatment group was tested.

Specified miR-6757-3p as the experimentally investigated arm.

Reframed apoptosis-related gene/protein changes as molecular markers that do not by themselves establish apoptosis.

Reframed Figure 5 as a time-dependent wound-closure assay and removed direct cellular-damage/protection interpretations.

Corrected AT9283 terminology and clarified its multi-target nature.

Aligned the ELISA Methods and Results and removed MDA/TAS from the described study endpoints.

Added explicit limitations concerning direct apoptosis assays, viability/proliferation assays, transfection efficiency, direct target validation, STR/mycoplasma testing, small replicate number, and independently determined qPCR amplification efficiencies.

Added a Data Availability Statement and clarified biological replicate reporting and qRT-PCR calculations.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors mainly examine the potential role of miR-6757 in regulating Aurora kinase signaling, apoptosis-related genes, and cell migration in A549 and BEAS-2B cells. The study provides some potentially useful observations, but several conclusions currently go beyond what are directly supported by the experimental design. In particular, the “co-treatment” concept and the proposed miR-6757–AURKA/B mechanism require further clarification. Therefore, I recommend that it undergo major revision.

The title of this manuscript emphasizes “Co-Treatment with miR-6757 and an Aurora Kinase Inhibitor,” suggesting that the biological interaction between miR-6757 modulation and Aurora kinase inhibition is a major focus of the study. However, the experimental groups described in the Results appear to consist of NC, miR-6757 inhibitor, miR-6757 mimic, and Aurora kinase inhibitor as separate treatments. I could not identify a clear miR-6757 mimic/inhibitor + AT9283 combination group in Figures 1–5. This point is important because the current design can show parallel effects of miR-6757 modulation and Aurora kinase inhibition, but it cannot establishs whether these interventions cooperate, antagonize each other, or act through the same pathway. The authors should either include and analyze genuine combination-treatment groups or revise the title, Abstract, and related conclusions to ensure the manuscript accurately reflects the experiments actually performed.
The authors report that miR-6757 inhibition alters AURKA/B expression and the expression of BAX, BCL2, CASP 3, CASP9, and MTOR, and interpret these changes as evidence that miR-6757 regulates Aurora kinase-related apoptosis and potentially PI3K/AKT/mTOR signaling. However, the study does not directly demonstrate that AURKA or AURKB is a functional mediator of the miR-6757 phenotype. In particular, the direct miRNA–target interaction is not tested, and the manuscript itself acknowledges the absence of a luciferase reporter assay, gene-specific silencing, Annexin V/PI assays, and Western blot validation. At a minimum, the conclusions should be toned down to distinguish association from causality. Ideally, the authors could strengthen the study with a focused rescue/epistasis experiment—for example, selective depletion of AURKA or AURKB in the context of miR-6757 inhibition, or restoration of Aurora kinase expression after miR-6757 mimic treatment—to determine whether the apoptosis/migration phenotype is actually Aurora kinase dependent.
The Methods refer to “AT9383” in one place, whereas the Abstract and general description refer to AT9283. These should be checked carefully and standardized. In addition, the scratch-assay section states that cells were “exposed to a concentration of 10 µg/mL,” but it is not clear which reagent this concentration refers to.
Figure 5 is described as a migration/time-course experiment, but the Results and figure legend repeatedly refer to “cellular damage,” a “protective effect,” and a “pro-apoptotic role.” A wound-healing assay primarily measures closure of a scratched area and can be affected by both migration and proliferation; it does not by itself demonstrate cellular damage or apoptosis. The terminology should therefore be changed to wound closure/migration unless additional viability or apoptosis measurements were performed at these time points.

The authors compare A549 lung cancer cells with BEAS-2B cells throughout the study, but some responses to miR-6757 modulation are quite different between the two cell lines. For example, Figure 1 shows marked differences in the magnitude and direction of AURKA/B expression changes between BEAS-2B and A549 cells. The authors should clarify whether BEAS-2B is intended primarily as a non-tumor lung epithelial control and discuss the cell-line-specific responses rather than combining them into a single general conclusion.
The manuscript states that the ELISA results are generally consistent with the gene-expression data. However, several protein measurements appear modest or statistically non-significant, particularly AURKB and BAX, while some treatment-associated changes are more evident for CASP3 and BCL 2. The text should clearly distinguish statistically supported findings from trends and avoid implying uniform agreement between mRNA and protein measurements.

Author Response

Comment 1

Reviewer comment: The title emphasizes “Co-Treatment with miR-6757 and an Aurora Kinase Inhibitor,” although the experimental groups appear to be NC, miR-6757 inhibitor, miR-6757 mimic, and Aurora kinase inhibitor as separate treatments. No clear miR-6757 + AT9283 combination group is shown. The authors should either perform genuine combination-treatment experiments or revise the title, Abstract, and conclusions to reflect the experiments actually performed.

Response: We agree with the reviewer. The study did not include a simultaneous miR-6757 mimic/inhibitor + AT9283 combination-treatment group. Therefore, the original use of the term “co-treatment” could imply an experimental interaction that was not directly tested. We have removed this terminology and revised the manuscript so that the findings are presented as separate effects of miR-6757 modulation and AT9283 treatment. We have also avoided claims regarding cooperation, antagonism, or pathway convergence between the two interventions.

Revision made in the manuscript: The title was revised to: “In vitro effects of miR-6757 modulation and AT9283 treatment on AURKA/AURKB expression and apoptosis-related responses in A549 and BEAS-2B cells.” The Abstract was revised to describe an association between miR-6757 modulation, Aurora kinase expression, and apoptosis-related responses rather than co-treatment. The Conclusion was also revised to state that the study provides preliminary in vitro evidence of an association and does not establish a causal signaling mechanism. These changes are reflected throughout the manuscript.

Comment 2

Reviewer comment: The manuscript interprets changes in AURKA/B, BAX, BCL2, CASP3, CASP9, and MTOR as evidence of regulation of Aurora kinase-related apoptosis and potentially PI3K/AKT/mTOR signaling, although no direct miRNA–target interaction or functional mediator experiment was performed. The reviewer recommends that conclusions be toned down and, ideally, strengthened by rescue/epistasis experiments.

Response: We appreciate this important point and agree that the original interpretation was too mechanistic. The present study does not establish that AURKA or AURKB is a functional mediator of the observed miR-6757-associated responses. Likewise, the available data do not demonstrate direct post-transcriptional targeting or establish a PI3K/AKT/mTOR-mediated mechanism. Because additional rescue/epistasis experiments were not performed, we have not presented these relationships as causal. Instead, the manuscript now consistently uses cautious terms such as “associated with,” “may,” and “suggest,” and explicitly distinguishes association from causality.

Revision made in the manuscript: The Discussion was revised to state that the observed AURKA/AURKB changes suggest an association with miR-6757 modulation but do not establish direct targeting. We explicitly acknowledge the absence of luciferase reporter assays, selective gene-silencing/rescue experiments, Western blot validation, and direct apoptosis assays. The manuscript now states that bioinformatic prediction and luciferase reporter assays would be required to determine whether AURKA/AURKB are direct targets. The Conclusion was also revised to emphasize that no direct molecular targeting or causal signaling mechanism was established and that future studies should include direct target validation, functional apoptosis assays, selective gene-silencing strategies, and in vivo models.

Comment 3

Reviewer comment: The Methods refer to “AT9383” in one place, whereas the manuscript otherwise refers to AT9283. The scratch-assay section also states that cells were exposed to “10 µg/mL” without clearly identifying the reagent.

Response: We agree. The compound name was checked and standardized as AT9283 throughout the manuscript. The concentration statement in the scratch assay was also clarified so that the treatment refers to the Aurora kinase inhibitor AT9283.

Revision made in the manuscript: The Materials and Methods section was corrected to use “AT9283” consistently. The previous typographical inconsistency (“AT9383”) was removed. In the Scratch-Induced Migration Assay section, the treatment was clarified as AT9283 at 10 µg/mL. The Results and figure descriptions were also standardized to use AT9283.

Comment 4

Reviewer comment: Figure 5 is described as a migration/time-course experiment, but the Results and legend refer to “cellular damage,” a “protective effect,” and a “pro-apoptotic role.” A wound-healing assay primarily measures wound closure and can be influenced by migration and proliferation; it does not by itself demonstrate cellular damage or apoptosis.

Response: We agree with the reviewer. We recognize that the scratch assay cannot independently establish cellular damage, protection, apoptosis, or a specific pro-apoptotic mechanism. The terminology has therefore been revised to focus on wound closure and wound-healing responses. We have also explicitly acknowledged that wound closure can reflect both migratory and proliferative processes and that the assay should not be interpreted as a direct apoptosis or viability assay.

Revision made in the manuscript: The Results section was revised to describe Figure 5 in terms of “wound closure,” “wound-healing response,” and “time-dependent changes in wound closure.” Statements implying direct cellular damage or a protective/pro-apoptotic effect were removed or qualified. The Discussion now explicitly states that the scratch assay does not allow direct conclusions regarding cellular damage, protection, or apoptosis. Figure 5 was also revised so that its legend accurately describes the wound-closure experiment and the data presentation.

Comment 5

Reviewer comment: The authors compare A549 lung cancer cells with BEAS-2B cells, but some responses differ substantially between the two cell lines. The authors should clarify whether BEAS-2B is primarily a non-tumor lung epithelial control and discuss cell-line-specific responses rather than combining them into a single general conclusion.

Response: We agree. BEAS-2B cells were included as a non-tumorigenic bronchial epithelial reference/control model, rather than as a second cancer model. We recognize that the responses to miR-6757 modulation were not identical between BEAS-2B and A549 cells. These differences may reflect cell-line-specific biological context and should not be interpreted as evidence of a universal response. We have therefore revised the interpretation to avoid overgeneralization and have discussed the cell-line-specific nature of the findings.

Revision made in the manuscript: The manuscript now clearly describes BEAS-2B as a non-tumorigenic bronchial epithelial control/reference model. The Discussion was revised to acknowledge differences between A549 and BEAS-2B responses and to emphasize that the effects of miR-6757 modulation may be context-dependent. We also avoided interpreting the BEAS-2B findings as establishing cancer specificity or a therapeutic window. The Conclusion was written more cautiously so that the observations are not generalized across cell types.

Comment 6

Reviewer comment: The manuscript states that ELISA results are generally consistent with gene-expression data, but several protein measurements are modest or statistically non-significant, particularly AURKB and BAX, whereas CASP3 and BCL2 show clearer changes. The text should distinguish statistically supported findings from trends and avoid implying uniform agreement between mRNA and protein measurements.

Response: We agree with the reviewer. The original wording overstated the degree of concordance between transcriptional and protein-level findings. We have revised the Results and Discussion to distinguish statistically significant changes from trends and to acknowledge that not all protein-level measurements followed the corresponding mRNA changes with statistical significance. The revised text now describes the protein findings as “partially consistent” rather than uniformly concordant.

Revision made in the manuscript: The Results section was revised to state that AURKA/AURKB and apoptosis-related protein levels showed patterns that were only partially consistent with the corresponding mRNA findings, and that not all changes reached statistical significance. The Discussion was similarly revised to avoid implying uniform agreement between mRNA and protein data. The manuscript now emphasizes the specific statistically supported findings and treats non-significant observations as trends rather than definitive effects.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and suggestions for authors are presented in biomedicines 4534985 REVIEW REPORT V2.

Comments for author File: Comments.pdf

Author Response

Comment 1. The observed apoptosis-related and migration findings appear inconsistent with the conclusion that miR-6757-3p acts as a tumor-suppressive miRNA.

Response: We thank the Reviewer for highlighting this important interpretative issue. We agree that the observed changes do not support a definitive classification of miR-6757-3p as either a tumor-suppressive or oncogenic miRNA. The revised manuscript therefore avoids assigning a specific tumor-suppressive or oncogenic role to miR-6757-3p. Instead, the findings are described as associations between miR-6757-3p modulation and changes in Aurora kinase expression, apoptosis-related molecular markers, and wound closure. We have revised the Discussion and Conclusions to emphasize that these responses may be context-dependent and that their functional significance remains to be established.

Comment 2. The proposed mechanism involving AURKA/AURKB signaling is internally inconsistent, and the findings do not establish a causal relationship.

Response: We agree that the observed responses following miR-6757-3p modulation and AT9283 treatment do not establish a specific Aurora kinase-mediated apoptotic mechanism. We have revised the manuscript to use association-based language rather than causal statements. We also clarify that AT9283 is a multi-target kinase inhibitor and that the molecular changes observed following its treatment cannot be attributed exclusively to Aurora kinase inhibition. The revised Discussion and Conclusions explicitly state that the present results do not establish a causal signaling mechanism.

Comment 3. There is no direct evidence that AURKA or AURKB is a direct target of miR-6757-3p.

Response: We agree with the Reviewer that the present study does not demonstrate direct binding of miR-6757-3p to the 3′ untranslated regions (3′UTRs) of AURKA or AURKB. We have revised the manuscript to avoid describing these genes as validated direct targets of miR-6757-3p. The observed expression changes are now interpreted as associations rather than evidence of direct post-transcriptional regulation. We have also explicitly acknowledged the absence of luciferase reporter assays and indicated that bioinformatic target prediction and experimental validation using wild-type and seed-mutant 3′UTR reporter constructs will be required in future studies.

Comment 4. The original title implied co-treatment, although the experimental groups received individual treatments.

Response: We thank the Reviewer for this clarification. We have revised the title to remove the term “co-treatment.” The revised title accurately reflects the experimental design, in which cells were treated individually with the miR-6757-3p inhibitor, miR-6757-3p mimic, or AT9283. No combined miRNA–AT9283 treatment group was included.

Comment 5. The mature miRNA arm investigated should be specified consistently.

Response: We agree. The revised manuscript consistently identifies the investigated miRNA as miR-6757-3p and distinguishes this arm from miR-6757-5p when discussing previous literature. The Methods section specifies the miR-6757-3p mimic and inhibitor used in the experiments.

Comment 6. Direct apoptosis, cell viability, and proliferation assays were not performed.

Response: We acknowledge that gene and protein expression measurements alone cannot establish the occurrence of apoptosis or determine changes in cell viability or proliferation. Annexin V/PI analysis, direct caspase activity measurements, and dedicated cell viability or proliferation assays were not included in the present experimental design. Rather than making unsupported functional claims, we have revised the manuscript to describe the findings as changes in apoptosis-related molecular markers. We have also clarified that the scratch assay measures wound closure and cannot independently distinguish migration from effects associated with cell proliferation or viability. These limitations are now acknowledged in the Discussion, and the need for direct functional assays in future studies is emphasized.

Comment 7. Transfection efficiency was not adequately documented, and the miR-6757-3p expression results were unexpected.

Response: We thank the Reviewer for raising this methodological concern. We have expanded the Methods section to specify that A549 and BEAS-2B cells were transfected with the miR-6757-3p mimic or inhibitor using EcoTRAX transfection reagent, with 25 pmol of the respective oligonucleotide per well and assessment of miR-6757-3p expression 24 hours after transfection. Corresponding negative-control oligonucleotides were included.

We acknowledge that an independent functional transfection-efficiency assay was not performed. Moreover, the measured miR-6757-3p expression did not consistently change in the expected direction following inhibitor or mimic treatment, and AT9283 treatment was associated with an unexpected increase in miR-6757-3p expression. These observations are now explicitly described in the Results and Discussion. We do not interpret the expression measurements as definitive confirmation of successful functional knockdown or overexpression. The unexpected expression pattern remains unresolved and is identified as an important limitation requiring further investigation.

Comment 8. The Methods and Results sections were inconsistent regarding ELISA and oxidative-stress measurements.

Response: We thank the Reviewer for identifying this inconsistency. We have revised the manuscript to improve the description and interpretation of the measured endpoints and to distinguish protein-expression analyses from wound-healing measurements. The revised text avoids interpreting wound closure as direct evidence of cellular damage or apoptosis. We acknowledge that MDA and total antioxidant status (TAS) measurements require clear and consistent reporting wherever these assays are retained in the manuscript. The final Methods, Results, figures, and supplementary information should correspond exactly to the experiments and data included in the revised submission.

Comment 9. The protein-level findings were described as more consistent and statistically significant than the figures support.

Response: We agree that the protein-level results should not be overstated. We have revised the Results and Discussion to clarify that the ELISA findings were only partially concordant with the corresponding gene-expression results and that not all comparisons reached statistical significance. In particular, the interpretation of AURKB and BAX protein measurements has been moderated in accordance with the comparisons shown in the figures. The revised manuscript distinguishes observed trends from statistically significant differences and avoids claiming uniform confirmation of the mRNA findings at the protein level.

Comment 10. The qPCR methodology and statistical reporting require improvement in accordance with MIQE recommendations.

Response: We appreciate the Reviewer’s detailed methodological comments. The revised manuscript clarifies the use of GAPDH for mRNA normalization and RNU6B_13 for miR-6757-3p normalization, the use of three independent biological replicates, and the calculation of relative expression using the 2⁻ΔΔCt method. We also acknowledge that amplification efficiencies were not independently determined and that only one reference gene was used for mRNA normalization. These limitations are now stated explicitly.

We recognize that additional validation, including amplification-efficiency assessment, evaluation of reference-gene stability, and reporting of raw or summary Ct/ΔCt values, would strengthen compliance with MIQE recommendations. We have not represented these additional validations as completed where they were not performed. The findings are therefore interpreted cautiously, particularly given the small number of biological replicates and the magnitude of some observed fold changes.

Reviewer 3 Report

Comments and Suggestions for Authors The authors have addressed the main concerns raised in the previous review. The revised title now reflects the experimental design, and the manuscript no longer implies a combination-treatment strategy that was not tested. The authors have also appropriately moderated their interpretation of the miR-6757-AURKA/AURKB relationship, clearly acknowledging the lack of direct target validation and functional apoptosis assays. In addition, the AT9283 nomenclature and scratch-assay conditions have been clarified; the wound-healing data are now described more appropriately; and the cell-line-specific responses, as well as the partial agreement between mRNA and protein measurements, are discussed more carefully. Overall, the revised manuscript is substantially clearer, and the conclusions are now better aligned with the available data. I have no further major concerns and consider the manuscript suitable for publication.

Author Response

Comment 1. The observed apoptosis-related and migration findings appear inconsistent with the conclusion that miR-6757-3p acts as a tumor-suppressive miRNA.

Comment 2. The proposed mechanism involving AURKA/AURKB signaling is internally inconsistent, and the findings do not establish a causal relationship.

Comment 3. There is no direct evidence that AURKA or AURKB is a direct target of miR-6757-3p.

Comment 4. The original title implied co-treatment, although the experimental groups received individual treatments.

Comment 5. The mature miRNA arm investigated should be specified consistently.

Comment 6. Direct apoptosis, cell viability, and proliferation assays were not performed.

Comment 7. Transfection efficiency was not adequately documented, and the miR-6757-3p expression results were unexpected.

Comment 8. The Methods and Results sections were inconsistent regarding ELISA and oxidative-stress measurements.

Comment 9. The protein-level findings were described as more consistent and statistically significant than the figures support.

Comment 10. The qPCR methodology and statistical reporting require improvement in accordance with MIQE recommendations.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors are presented in biomedicines-4534985 REVIEW REPORT V3.pdf.

Comments for author File: Comments.pdf

Author Response

Comment 1. Validation of miR-6757-3p transfection efficiency

Response: We thank the reviewer for this important comment. We acknowledge that the efficiency of miR-6757-3p mimic and inhibitor transfection was not independently validated using a dedicated transfection-efficiency assay. Although miR-6757-3p expression was assessed by miRNA-specific qRT-PCR, the observed expression patterns did not consistently demonstrate the expected direction of modulation. Therefore, successful functional overexpression or inhibition cannot be conclusively established from the available data. We have revised the manuscript to avoid interpreting the observed molecular changes as definitive evidence of successful miR-6757-3p modulation. These findings are now interpreted cautiously, and the absence of independent transfection-efficiency validation is acknowledged as a limitation.

Revision made in the manuscript: The Methods section was revised to clarify the absence of an independent transfection-efficiency assay (Lines 144–148). The Results section was revised to describe the inconsistent miR-6757-3p expression patterns and their implications (Lines 332-343) and (Lines 385-390)

Comment 2. Differences between gene and protein expression

Response: We thank the reviewer for this valuable comment. We agree that changes in gene expression do not necessarily correspond to changes at the protein level. Therefore, we have revised the manuscript to avoid presenting the protein results as direct validation of the gene-expression findings. Differences between mRNA and protein expression are interpreted cautiously, considering that transcriptional and post-transcriptional regulation may contribute to these discrepancies. We have also acknowledged the absence of Western blot analysis as a limitation and emphasized the need for further studies to clarify these findings.

Revision made in the manuscript: The Results section was revised to distinguish protein-level changes from the corresponding mRNA findings and to clarify that not all changes reached statistical significance (Lines 268–approximately 285). The Discussion was revised to avoid implying uniform agreement between gene and protein expression results (Lines 400–406).

Comment 3. Causal mechanisms and PI3K/AKT/mTOR signaling

Response: We thank the reviewer for this important comment. We agree that the original interpretation could imply causal relationships that were not directly investigated. Our experiments do not establish AURKA or AURKB as functional mediators of the responses associated with miR-6757-3p modulation, nor do they demonstrate direct miRNA–target interactions or a PI3K/AKT/mTOR-mediated mechanism. Accordingly, we have revised the Discussion and Conclusion to distinguish observed molecular associations from causal mechanisms. Changes in MTOR expression are not interpreted as evidence of pathway activation, and apoptosis-related gene-expression changes are not presented as direct confirmation of apoptosis. The absence of rescue/epistasis experiments and direct target-validation assays is acknowledged.

Revision made in the manuscript: The Discussion was revised to interpret AURKA/AURKB expression changes as associations rather than evidence of direct regulation (Lines 404–410). The Conclusion was revised to clarify that direct molecular targeting and a causal signaling mechanism have not been established (Lines 443–452).

Comment 4. Interpretation of apoptosis-related findings

Response: We thank the reviewer for this valuable comment. We agree that changes in apoptosis-related marker expression cannot independently demonstrate apoptotic cell death. We have revised the Abstract, Results, Discussion, and Conclusion to describe the findings as changes in apoptosis-related markers rather than evidence that apoptosis was directly induced or increased. These revisions ensure that the interpretation remains within the scope of the available experimental evidence.

Revision made in the manuscript: The Abstract was revised to describe changes in apoptosis-related markers (Lines 22–32). The Results section was revised to report changes in BAX, BCL2, CASP3, CASP9, and MTOR expression without treating them as direct proof of apoptosis (Lines 236–252). The Discussion and Conclusion were also revised to use cautious, association-based language (Lines 419–423 and 443–452).

Comment 5. Absence of functional apoptosis assays

Response: We thank the reviewer for this important comment. We agree that changes in apoptosis-related gene and protein expression alone do not directly demonstrate apoptotic cell death. As no additional functional assays were performed, we have acknowledged the absence of cell-viability, Annexin V/PI, caspase-activity, cell-cycle, and Western blot analyses as a limitation. We have also revised the interpretation of the findings to avoid claims of confirmed apoptosis induction.

Revision made in the manuscript: The Results section was revised to describe molecular changes rather than confirmed apoptotic cell death (Lines 236–252 and 268–285). The Discussion and Conclusion were revised to clarify that the observed findings do not establish apoptosis or a specific apoptotic mechanism (Lines 419–423 and 443–452).

Comment 6. Wound closure assay and cell migration

Response: We thank the reviewer for this valuable comment. We agree that the wound closure assay cannot distinguish the effects of cell migration from those of cell proliferation. We have revised the manuscript to describe the findings as “wound closure” or “wound-healing response” rather than direct evidence of altered cell migration. We have also clarified that cell proliferation was not independently assessed and that the results should therefore be interpreted cautiously, particularly following AT9283 treatment.

Revision made in the manuscript: The Methods section describes the wound-closure procedure and its measurement over time (Lines 190–199). The Discussion was revised to focus on wound closure and to avoid conclusions regarding direct cellular damage, protection, or apoptosis (Lines 411–423).

Comment 7. Reliability of RT-qPCR results

Response: We thank the reviewer for this important comment. We clarified that the qRT-PCR analyses were performed using three independent biological replicates (n = 3). We also acknowledged that amplification efficiencies were not independently determined and that miR-6757-3p expression did not consistently change in the expected direction following inhibitor or mimic treatment. Therefore, the results cannot be considered definitive evidence of successful functional knockdown or overexpression. These methodological limitations and the need for further validation are now explicitly acknowledged.

Revision made in the manuscript: The Methods section was revised to report the biological replicates and to acknowledge that amplification efficiencies were not independently determined (Lines 164–176). The Results section was revised to describe the observed miR-6757-3p expression patterns and their limitations (Lines 247–252).

Comment 8. Direct targeting of AURKA/AURKB by miR-6757-3p

Response: We thank the reviewer for this valuable comment. We agree that the observed changes in AURKA and AURKB mRNA expression following miR-6757-3p mimic and inhibitor transfection do not establish a direct target relationship. Accordingly, we have revised the Discussion and Conclusion to avoid presenting these genes as confirmed direct targets of miR-6757-3p. We have also acknowledged the absence of seed-site validation and luciferase reporter assays as a limitation. Further bioinformatic and experimental studies are needed to determine whether these genes are direct targets of miR-6757-3p.

Revision made in the manuscript: The Discussion was revised to state that the observed expression changes indicate an association rather than confirmed direct targeting, and that bioinformatic prediction and luciferase reporter assays are required for further validation (Lines 404–408). The Conclusion was revised to clarify that direct molecular targeting has not been established (Lines 448–452).

Comment 9. Responses observed in BEAS-2B cells

Response: We thank the reviewer for this important comment. We agree that the observed changes in BEAS-2B cells limit the interpretation of the findings as cancer-specific effects. BEAS-2B cells are described as a non-tumorigenic bronchial epithelial reference model, and differences between the two cell lines are interpreted cautiously. We have revised the Discussion and Conclusion to avoid strong claims regarding cancer-specific mechanisms, biomarkers, therapeutic targets, or potential cancer treatments.

Revision made in the manuscript: The Introduction clarifies the use of BEAS-2B cells as a non-tumorigenic comparator (Lines 104–113). The Discussion acknowledges that the findings do not establish a tumor-suppressive or oncogenic role for miR-6757-3p and that the responses may be context-dependent (Lines 426–433). The Conclusion was revised to avoid claims of cancer specificity or therapeutic significance beyond the available evidence (Lines 443–452).

Comment 10. STR cell-line authentication and mycoplasma testing

Response: We thank the reviewer for highlighting the importance of cell-line authentication and mycoplasma testing. STR-based cell-line authentication and mycoplasma testing were not independently performed in this study. We acknowledge this as a limitation and have explicitly clarified the status of these tests in the revised manuscript.

Revision made in the manuscript: The Cell Culture section now explicitly states that STR profiling and mycoplasma testing were not independently performed (Lines 135–137). This limitation is also acknowledged in the Discussion (Lines 436–440).

We believe that these revisions improve the transparency of the methods and the consistency between the experimental findings and their interpretation. We thank the reviewer again for helping us strengthen the scientific rigor of the manuscript.