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All issues pointed by the reviewers were adequately addressed and revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
see "Additional comments" section
see "Additional comments" section
See "Additional comments" section
The quality of the manuscript has improved thanks to the changes made. I think it could be of interest to the readers and, in my opinion, it deserves priority to be published
The revised manuscript is improved and ready for publication. The authors have addressed previous comments thoroughly, enhancing the clarity, flow, and scientific depth of the text.
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Overall, the manuscript presents novel insights and meets the standards for acceptance.
Please address the concerns of all reviewers and amend the manuscript accordingly.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
**PeerJ Staff Note:** Please ensure that all review and editorial comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
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I read with great interest the Manuscript titled " Mrs2 and mitochondrial gene networks in endometrial cancer: Mechanisms, biomarkers, and therapeutic implications”-
Although the manuscript can be considered of good quality, I would suggest the following recommendations:
- I suggest a round of language revision to correct a few typos and improve readability.
- In recent years, significant advancements have been made in the molecular and genomic profiling of endometrial cancer. Given the results and focus of this study, I recommend that the authors include references to recent evidence in this area, particularly regarding the potential prognostic significance of novel factors and molecular targets. I would be glad if the authors discuss this important point, referring to: 37454351 and 35008194.
Because of these reasons, the article should be revised and completed. Considering all these points, I think it could be of interest to the readers and, in my opinion, it deserves priority to be published after minor revisions.
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The manuscript needs substantial language editing to improve clarity and eliminate grammatical errors.
The manuscript is too lengthy and repetitive, particularly in the Discussion.
The selection of the 16 mitochondrial genes and their prioritization for downstream analysis should be better justified.
The validation cohort size is too small; this limitation should be addressed.
There is no mention of ethical approval.
The claim that Mrs2 may serve as a therapeutic target is premature without in vivo validation.
The introduction should be extended and completed. I find it interesting to include a reference to the prognostic role of L1CAM in endometrial cancer (see PMID: 37454351).
**PeerJ Staff Note:** The PeerJ's policy is that any additional references suggested during peer review should only be included if the authors find them relevant and useful.
General Comments:
The study comprehensively investigated the correlation between endometrial cancer and several mitochondrial proteins (e.g., Mrs2) using bioinformatics and experimental approaches, including lactate detection, flow cytometry, immunofluorescence, CCK8 assays, and transwell migration assays. The work provides information on identifying potential biomarkers for detecting endometrial cancer.
However, some of the bioinformatics analyses and methods used in the paper have not been clearly described. Details are missing in the Figure legend and Methods. For instance, information on the source of datasets and critical parameters used is missing in most analyses. It is not sufficient for the reader to evaluate the work or replicate the experiment. It is strongly recommended to include this critical information in the revised manuscript. Also, the manuscript should be revised to present the data more clearly in the Results section and to ensure it is appropriately linked to the corresponding figure. Some of the data shown in the figure has not been described in the Results section, for example, Fig. 3D, E. Figures with multiple panels need to be more clearly described and referenced in the main text, rather than briefly referenced in a sentence (Line 140, Figure 4, 5).
Other Comments:
- Line 40-41, “superconducting magnesium channel magnesium transporter (Mrs2)” should be revised to the full name of MRS2, “Mitochondrial RNA Splicing 2 protein”.
- Line 41, MRS2 is a channel, not a transporter
- Line 45, “reservoir of cell suicide weapons” sounds awkward, please revise.
- Line 53, revise “corA/Mrs2/Alr1 protein super-family of Mg2+ transport proteins” to “corA/Mrs2/Alr1 super-family”
- Line 54, “C-terminal end” instead of ”cytoplasmic end”
- Line 64-66, Daw et al (PMID: 33035451) should be cited
- Figure 1C: An additional label was found in the chart. please check
1. The rationale of testing lactate level upon LPS stimulation in relation to cancer proliferation is unclear in the Result 2.4 section. Authors should explain more about it before directly going into the observations.
2. In the Method, details information for the Estimation of lactate content is heavily missing.
3. In Figure 5, what fluorescence signal is used to monitor MRS2 expression and localization?
1. In Table 1 and Figure 1C, patients were classified based on expression of MRS2 (High or Low). Authors should describe how the threshold for High expression or Low expression is defined.
2. In the result section 2.4, authors observed that lactate levels and ROS in KLE increased upon LPS stimulation, and suggested that lactate upregulates Mrs2 expression in KLE cells, which in turn promotes ROS production, facilitating tumor proliferation and invasion. However, the causal relationship between lactate, MRS2, ROS, and tumor proliferation is not demonstrated. At least MRS2 knockout cells should be used to test whether MRS2 expression is crucial for promoting tumor proliferation. Otherwise, the conclusion should be toned down. In addition, ROS has a dual role in cancer progression, as also mentioned by the authors in the Discussion, the level of ROS increased may not be the major factor causing enhanced cell migration.
1.1 Clarity and Professional English
• Minor issues: A few typographical errors (e.g., “ransport” instead of “transport” in line 56) and inconsistent use of articles (e.g., “the superconducting magnesium channel magnesium transporter” at line 41 is unclear). I recommend a careful copy‐edit to correct these minor slips and ensure uniform terminology (e.g., consistently “Mrs2” vs. “MRS2” in line 304).
1.2 Literature References and Background
• The rationale for focusing on lactate‐mediated regulation of Mrs2 could be better contextualized by referencing recent studies on tumor glycolysis and lactate signaling in EC (e.g., Warburg effect literature).
• Some key hypotheses (e.g., “Mrs2 and its related genes may serve as key regulatory genes for mitochondrial metabolic abnormalities in EC,” lines 80–82) would benefit from referencing prior functional studies or reviews on magnesium transporters in other cancers.
1.3 Structure, Figures, and Raw Data
• Raw data sharing: The authors note raw data availability in supplementary files, but the Data Availability Statement (“original contributions … are included in the article”) is vague. They should specify repository accession numbers for the raw sequencing counts, DESeq2 outputs, and bioinformatics scripts to meet PeerJ’s Data Sharing policy.
2.1 Rigor and Ethical Standards
• Concerns: There is no statement of ethical approval for use of human data (TCGA and GEO are public, but immunohistochemistry from HPA and any patient‐derived samples should reference IRB exemption or approval). Additionally, the use of LPS to simulate “lactate‐rich” conditions is unconventional—LPS is a bacterial endotoxin that activates TLR4; clarification is needed on why LPS was used rather than direct lactate supplementation.
2.2 Methods Detail and Reproducibility
• Reagents and vendors: While some are specified, e.g., “Affinity Biosciences” for Mrs2 antibody, catalog numbers for key reagents (antibodies, LPS source) should be provided.
• Statistical analysis: The criteria for choosing one‐way ANOVA vs. Wilcoxon signed‐rank test lack detail—e.g., normality testing.
• Bioinformatics scripts: A GitHub link or supplemental methods file with R scripts would greatly enhance reproducibility.
• The mechanistic link between Mg²⁺ transport by Mrs2 and ROS production is inferred but not directly tested (e.g., Mg²⁺ flux assays, Mrs2 knockdown/rescue). The authors should temper claims of causation and propose these as future directions.
• The in vitro experiments lack Mrs2 knockdown or overexpression controls to confirm specificity. Incorporating siRNA or CRISPR‐based manipulation of Mrs2 would strengthen causal inference.
The authors investigate the role of the mitochondrial magnesium transporter Mrs2 and its network of associated genes in the progression of endometrial cancer (EC). They ask three core questions: (1) How does Mrs2 expression correlate with clinical outcomes in EC? (2) What mitochondrial‐related genes interact functionally with Mrs2, and what is their prognostic significance? (3) By what mechanisms does Mrs2 influence EC cell behavior, immune infiltration, and drug sensitivity? Addressing these questions is important because EC prognosis for advanced or recurrent disease remains poor, and mitochondrial metabolism—particularly magnesium‐dependent processes—represents an underexplored avenue for biomarkers and therapy in EC .
To answer these questions, the authors combine:
• Bioinformatics analyses of TCGA‐UCEC and GEO datasets to identify differential expression of Mrs2 and its mitochondrial function–related genes, perform survival and ROC analyses, and correlate gene expression with immune cell infiltration and drug sensitivity.
• In vitro experiments in KLE EC cells, including lactate assays, ROS measurement by flow cytometry, CCK8 proliferation assays, Transwell migration assays, and immunofluorescence to assess Mrs2 expression and function under lactate stimulation.
• Transcription factor prediction (ChEA3) and protein‐level validation via the Human Protein Atlas.
They conclude that Mrs2 is upregulated in EC, correlates with poor differentiation and survival, and in a lactate‐rich environment, drives ROS production, proliferation, and invasion of EC cells. Three key Mrs2‐associated genes (MRPL15, MTHFD2, MTFR2) emerge as prognostic markers. Mrs2 expression also correlates with altered immune infiltration (e.g., increased CD4⁺ T cells, decreased NK cells) and predicts sensitivity to the IGF1R inhibitor BMS-754807. Overall, the study positions Mrs2 and its network as potential biomarkers and therapeutic targets in EC .
Overall, this study was well formatted. I have a few suggestions for the authors to improve their study:
1. Data Sharing: Deposit raw sequencing data, processed matrices, and analysis scripts in accessible repositories (e.g., GEO, GitHub) and update the Data Availability Statement accordingly.
2. Mechanistic Validation: Perform Mrs2 loss‐ and gain‐of‐function experiments (e.g., siRNA knockdown) to directly test its role in Mg²⁺ transport, ROS generation, and cell behavior.
3. Clarify LPS Use: Justify or replace LPS stimulation with direct lactate treatment to model the tumor microenvironment more accurately.
4. Copy‐Editing: Address minor typographical and consistency issues, and consider professional language editing to polish the manuscript.
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