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All the issues raised by the reviewers have now been addressed. The manuscript is now ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Gwyn Gould, a PeerJ Section Editor covering this Section #]
The authors have made all the corrections in the revised manuscript and provided satisfactory justifications.
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• Rationale for Selecting miR-597-3p in Ovarian Cancer
The manuscript lacks a clear justification for selecting miR-597-3p for investigation in ovarian cancer. The authors should provide a rationale supported by literature or preliminary data demonstrating the relevance of miR-597-3p in the pathogenesis or progression of ovarian cancer, particularly concerning its tumor-suppressive function.
• Target Identification Strategy for miR-597-3p
Typically, to identify the functional targets of a microRNA—especially one with tumor-suppressive properties like miR-597-3p—overexpression experiments followed by RNA analysis (e.g., RNA sequencing) are standard. In this study, however, the authors rely solely on in silico predictions from TargetScan. For more robust target validation, the inclusion of RNA-seq data or additional experimental validation (Western blot for predicted targets) is strongly recommended.
• MACC1-Mediated Regulation of MMP-2 and MMP-9
The authors' claim that MACC1-driven networks downregulate MMP-2 and MMP-9 to reduce invasiveness in ovarian cancer is not directly supported by functional evidence. While relative expression data are provided, the conclusion would be significantly strengthened by the inclusion of Western blot analyses to confirm the protein-level changes of MMP-2 and MMP-9 following MACC1 modulation.
• Clarification of Apoptosis Experiment Protocol
The experimental protocol describing the apoptotic effect of miR-597-3p in SKOV-3 cells lacks critical detail. Specifically, the incubation period after transfection with the miR-597-3p mimic is not stated. Please clarify the duration between transfection and downstream apoptosis assays, as this is essential for reproducibility.
• Lack of Explanation Regarding Bax and Bcl2L12
Figure 5A presents data on Bax and Bcl2L12, yet their roles concerning miR-597-3p in SKOV-3 cells and ovarian cancer are not clearly explained. As key regulators of apoptosis, Bax is pro-apoptotic, and Bcl2L12 is anti-apoptotic. The authors should briefly elaborate on how these proteins are modulated by miR-597-3p and/or MACC1, and how this contributes to the apoptotic response. Adding this context would enhance the clarity and relevance of the findings.
Some experimental procedures, such as the apoptosis assay protocol, require further clarification. Precise details, such as incubation times, concentrations, and assay conditions, should be provided to ensure reproducibility and transparency.
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The article is well written and presents a relevant study. However, to improve the clarity, reproducibility, and scientific accuracy of the manuscript, the following revisions are required:
1. Materials and Methods: Ensure that all subsections provide sufficient detail to allow replication of the experiments. Descriptions should be precise and complete.
2. Transfection and transwell assay: These sections currently lack critical experimental details. Please include specific concentrations, incubation times, and step-by-step procedures to enhance reproducibility.
3. AO/EB staining (Line 202): The statement that “cells were lysed” appears to be incorrect or misleading in the context of AO/EB staining. Please revise this sentence to accurately reflect the procedure performed.
4. Invasion assay: Clarify the method used to calculate the percentage of invasion. The formula or calculation approach should be explicitly stated.
5. Fluorescence/luminescence quantification: The methodology for capturing and quantifying luciferase activity is insufficiently described. Specify the equipment used, the detection method, and any software employed for data analysis.
6. Lines 245, 255, 324: These sentences are unclear and should be rewritten for clarity and grammatical accuracy.
7. AO/EB staining cannot reliably distinguish between apoptosis and necrosis; it only indicates general cell death. The interpretation of results should be revised accordingly unless supported by additional apoptotic markers.
8. Figures 5B/C and 6B/C: The explanations for these panels appear to be swapped. Please correct the figure legends. Additionally, the statement regarding MMP2 and MMP9 being pro- or anti-apoptotic is overly generalized. These roles are context-dependent and should be revised to reflect the variability based on cell type and experimental conditions.
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