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Authors have addressed all of the reviewers' comments and the manuscript is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
Please address remaining comments of reviewer 2.
The authors have addressed most of the points raised in the previous review, and the revised manuscript is clearer and includes additional details that enhance the overall quality. The inclusion of bioinformatic details and updates to the figures are noted improvements.
However, several critical gaps remain that need to be considered:
Experimental Validation: Although the authors have acknowledged the lack of experimental validation regarding CYTOR’s role in resistant melanoma cell lines, this is still a notable limitation. The absence of experimental data in this area continues to reduce the strength of the manuscript’s conclusions. I recommend the authors clearly state in the discussion that this gap is acknowledged and will be addressed in future research.
Controls and Experimental Design: The authors have mentioned the absence of experiments involving normal melanocytes, and while this limitation is acknowledged, it would be helpful for the authors to emphasize this as a priority for future studies to provide a more complete context for their findings.
Bioinformatics Transparency: The additional details regarding bioinformatic analysis are appreciated. However, further clarification on the datasets used, as well as the rationale for choosing specific thresholds and parameters, would improve the transparency and robustness of the methodology.
While the manuscript has improved, addressing these remaining gaps would enhance its overall rigor. Therefore, I recommend it for publication with these points considered.
no comment
no comment
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript.
Interestingly, the authors addressed the role of CYTOR in treatment resistance of melanoma, which is of great interest. However, their response was insufficient, as they simply stated: "Due to the experimental limitations of our lab and the different focus of this study design, we did not cover resistant melanoma cell lines. We are willing to assess these cell lines in future studies."
Authors are suggested to address remaining/ new comments made by the reviewers. In addition, please provide 'full-length, uncropped Western blots showing selected bands and their sizes' as additional file for reviewer's assessment.
The authors have made significant improvements in the manuscript’s clarity and contextual depth by addressing treatment resistance in melanoma. The expanded introduction now includes a robust discussion of resistance mechanisms, such as MAPK/ERK pathway reactivation and the roles of specific lncRNAs, which enhances the clinical relevance of their work. Additionally, they clarified the data source for Figure 1D, ensuring transparency and adherence to ethical research standards.
The updates to Figure 1A, including group labels within the plot, improve accessibility for readers. Similarly, the inclusion of details about the bioinformatics tools (Starbase and TargetScan) used for molecular interaction predictions strengthens the manuscript’s methodological reporting. However, additional details about the bioinformatics workflow (e.g., thresholds or parameters) would further enhance transparency.
The authors' response to the clinical translation potential of CYTOR is satisfactory, with a relevant discussion of its role in other cancers. Including a brief discussion of RNA-based therapeutic strategies could enhance this section further.
The manuscript’s focus on CYTOR as a potential biomarker is well-supported by the study design. However, the lack of experimental validation of CYTOR’s role in treatment resistance in melanoma remains a limitation. The authors acknowledge the importance of studying CYTOR in resistant melanoma cell lines but cite experimental limitations as the reason for not including this aspect. While their willingness to explore this in future studies is noted, this omission leaves a critical gap in the manuscript’s experimental rigor.
The addition of bioinformatic analyses is a step forward, but the details provided are minimal. Expanding the methodology to include specifics about the analysis (e.g., thresholds, datasets, or parameters used) would significantly improve the experimental robustness and allow readers to evaluate the findings more thoroughly.
The findings presented in the manuscript are consistent with the current understanding of melanoma biology and highlight CYTOR as a potential biomarker. The authors effectively address the concern about providing context for treatment resistance, reinforcing the clinical relevance of their work.
However, the absence of experimental data on CYTOR’s role in resistant melanoma models limits the study’s ability to substantiate its claims fully. While the authors have cited studies demonstrating CYTOR’s involvement in resistance mechanisms in other cancers, direct validation in melanoma would greatly strengthen the manuscript's impact. The inclusion of such experiments or even preliminary data would enhance the validity of the conclusions drawn.
Overall, the manuscript represents an important contribution to melanoma research but would benefit from further methodological and experimental additions to address remaining gaps.
The manuscript is well written, and contains relevant references, and the revised introduction is appropriate.
The manuscript is well set out with appropriate tables and figures.
I have no further comments on the manuscript.
The authors have address the points raised in the review, and as such I have no further comments to add.
The authors have address the points raised in the review, and as such I have no further comments to add.
This manuscript investigates the role of the long non-coding RNA CYTOR in melanoma progression through its regulation of miR-485-5p and glucose-6-phosphate isomerase (GPI). Using in vitro and in vivo models, the study demonstrates that CYTOR is significantly upregulated in melanoma tissues and cell lines. It promotes cell proliferation, migration, and invasion, with potential implications as a therapeutic target or biomarker. The authors have addressed most previous reviewer comments, improving the clarity and scope of their work.
General Comments
The revisions significantly improve the manuscript, particularly with the inclusion of treatment resistance in melanoma and added methodological details. However, some points still require further refinement to meet publication standards.
Comments:
Basic Reporting
Language and Terminology:
The authors have addressed prior concerns about terminology (e.g., replacing "white populations" with "Caucasians"). No further revisions are necessary regarding terminology.
Ensure consistency in italicizing Latin terms (e.g., in vivo, in vitro) throughout the manuscript.
Figures:
Scale bars are now included in scratch assay and transwell images. However, ensure consistent labeling across all figures for improved clarity (e.g., Figures 2C, 3C, 3D).
Figures 1 and 3 would benefit from annotated labels indicating experimental groups directly on the plots for reader accessibility.
References:
While the reference list has improved, minor inconsistencies remain in formatting (e.g., some entries mix journal abbreviations and full names). Ensure all references include volume and page numbers for completeness.
Experimental Design
Control Experiments:
Normal Melanocytes: While the authors acknowledge limitations in not assessing CYTOR inhibition in normal melanocytes (e.g., PIG1), this remains a critical gap. Clearly state this limitation in the discussion and suggest it as a priority for future research.
Bioinformatics Details:
The inclusion of GEPIA2 and Starbase for bioinformatic analyses strengthens the study. Ensure these methods are described thoroughly in the main text and supplementary files, including specific thresholds used in analyses (e.g., |Log2FC|, p-values).
Validation of CCK-8 Results:
Address concerns about CCK-8 absorbance potentially reflecting residual reagent from prior incubations. While the authors clarified the assay protocol, additional validation (e.g., protein quantification) would improve confidence in the findings.
Xenograft Model:
The xenograft experiment is well-detailed. However, explicitly describe the statistical criteria for assessing tumor growth differences between experimental groups.
Western Blot Quality:
Concerns about blot quality and antibody specificity persist. Full-length, uncropped Western blots should be included in the supplementary materials to ensure transparency and address concerns regarding band selection.
Therapeutic Potential of CYTOR:
The authors note the absence of drugs targeting CYTOR in clinical trials but should elaborate on how CYTOR might be targeted therapeutically (e.g., RNA interference, small molecule inhibitors). Provide examples of similar strategies used for other lncRNAs.
Clinical Relevance:
Highlight potential applications of CYTOR as a diagnostic or prognostic biomarker in melanoma, particularly in cases of therapeutic resistance.
Clarity and Conciseness:
While the revised manuscript is improved, ensure seamless integration of new sections (e.g., treatment resistance). Avoid redundancy, especially in the introduction and discussion.
Discussion:
Expand on future directions, particularly how CYTOR could be incorporated into therapeutic strategies or used as a biomarker in drug resistance models.
This manuscript needs a thorough revision specially results and discussion part. Please address comments of all reviewers and provide responses in a point wise manner.
[# PeerJ Staff Note: PeerJ can provide language editing services if you wish - please contact us at [email protected] for pricing (be sure to provide your manuscript number and title). Your revision deadline is always extended while you undergo language editing. #]
The manuscript is written in clear and unambiguous English. Few typos can be found.
The references are up to date and are clearly connected to the topic of the manuscript.
The manuscript follows the professional article structure, figures are clear and well-prepared.
Supplementary data contains original figures to check the possibility of manipulations.
The results support the hypothesis and are discussed in the discussion section.
The design is well prepared, experimental data described in materials and methods are sufficient to repeat the experiments, the sequences of all si-RNAs and primers have been submitted.
I would add a subchapter of bioinformatics analysis regarding mir-485-5p targets, and the use of public bioinformatics tools such as GEPIA2 or preparation of KM plots. Also, the description of used cell lines (i.e. mutation status or type of melanoma) would be beneficial. The authors should also check the influence of CYTOR inhibition on normal melanocyte cell lines.
The results support the hypothesis and are discussed in the discussion section. Statistical data is flawless. The conclusions are interesting, making CYTOR a potential target in melanoma treatment.
No comments
Lu et al. investigated the role of the long non-coding RNA CYTOR in melanoma, presenting a well-written and thoughtfully designed study. The topic is both timely and significant, given the pressing need to identify novel therapeutic targets for this aggressive cancer. While the manuscript is strong overall, I recommend a few modifications that could enhance the discussion and the impact of the findings:
1. Introduction: The authors highlight the progress made in melanoma treatment in recent years due to the discovery of new biomarkers. However, they do not address the critical issue of treatment resistance, which remains a major hurdle in melanoma therapy. Including a discussion of resistance development would provide important context and underscore the clinical relevance of CYTOR.
2. Figure 1A: While the legend is clear, the plot itself would benefit from including labels for the groups directly within the figure. This would make the data more accessible to readers.
3. Figure 1D: The source of the data presented in this figure is not immediately apparent. Please clarify where this data was derived from, whether from a specific dataset, experiment, or prior study.
4. Bioinformatic Analyses (Sections 3.4 and 3.5): The authors state that bioinformatic analyses guided their focus on specific targets. However, these analyses are neither included nor cited. Providing details or references for these analyses would strengthen the methodological rigor of the study and allow readers to evaluate the evidence supporting the selected targets.
5. Clinical Translation Potential: The manuscript discusses CYTOR as a promising biomarker, but it does not address its translational potential. Are there any drugs targeting CYTOR, or is it the focus of ongoing clinical trials? Including such information would enhance the relevance of the study for clinical applications.
6. Resistance to Targeted Therapy: As mentioned earlier, treatment resistance is a significant issue in melanoma management. It would be highly valuable for the authors to test CYTOR modulation in resistant melanoma cell lines and to measure CYTOR levels in these models. Such experiments would provide critical insights into its role in overcoming therapeutic resistance.
By addressing these points, the authors can substantially improve the manuscript’s impact and relevance, ensuring it resonates with a broader audience in melanoma research and clinical oncology.
no comment
no comment
The standard of English used in the manuscript is fine. However, it is advisable on L33 to replace "white populations" with "Caucasians" which is the more appropriate term to use. Latin words such as "in vivo" an "in vitro" should be written in italics. L134 replace "same-sex" with "same gender"
The sentence L40 "Melanoma treatment....." in what way and it markedly improved, its detection or treatment or both, how has this taken place?
References need to be revised, as a mixture of journal abbreviations and full names have been used throughout, please be consistent and use one or the other, but not both. Please list volume and page numbers for all references. See L416, 421 etc for example.
Images of scratch assays and Transwell migration should show a scale bar. See Fig 2C, 3C, 3D etc.
Raw data has been attached as supplementary data.
The experiments performed by the authors are logical. They show that CYTOR expression is elevated in melanoma tissues and cell lines. CYTOR was shown to stimulate cell proliferation, migration and invasion. They also show that miR-485-5p binds to CYTOR and that GPI was a direct target of this lncRNA. they confirmed their findings using melanoma cell lines and with tumours grown in vivo.
Section 2.5 L108. I take it the same wells were treated with CCK-8 every 24 h then the abs read on plate reader, is that correct? For how long was CCK-8 left on the cells? was it washed off and the cells given fresh media? How can you be certain that increased abs is not due to the retention of CCK-8 after each incubation over this 96 h. Please comment on what you did here. Were parallel experiments performed to show increased levels of cell protein each 24 period?
Section 2.6. L116. What is the size of the pipette tip? 200 µL, 1 mL etc?
L117 what do you mean addition 24 h incubation period in what media. This detail is missing.
Section 2.9. What are the species of the antibodies used in this study, are they mouse monoclonals or rabbit polyclonal or something else? No information is given. You do not block the PVDF membrane with skim milk, but with skim milk powder, there is a difference, so please state precisely what was used.
The original Western blot images are of great concern. These blots show multiple bands for the antibodies which are of great concern, and as the authors do not state if these are mAb or pAb, one is led to believe that these Ab are at best extremely poor pAb. The authors need to state the origin of these antibodies as they selectively have cut one small part of the gel which has multiple bands visible indicating that these antibodies are of extremely poor quality especially a-tubulin.
I did find odd that the legends for the figures are not opposite the actual figure but in the main part of the text. It made the results section much larger than it is. In fact, there is a lack of data in the results section, in fact no numbers to show differences in outcome for each assay is shown. Please state key findings rather than just show a descriptive results section eg. L174 the authors should state that the expression in CYTOR was highest in A375 cells which was 6 times that seen in PIG1 melanocyte cels. The authors should state that the minimum increase in the different melanoma cell lines is 2.x-fold that seen in the melanocyte cells. There is a lack of hard data shown in the text of the results section.
Can the authors explain why in Fig 2 and 3 the melanoma cells used are different? Was there a reason for this? Different cells were used in Fig 6.
In Fig 7 what cell line was transfected and allowed to grow in the mice? It is stated on L132 but not in section 3.7 where it should be stated as well. Please correct.
In the discussion, please reference the statement on L312 "GPI is a .....", you state that GPI is recognised to modulate cell proliferation, but you do not reference this statement. Much of L315-324 discusses other tumour cells but not melanoma, what is known about GPI in melanoma cells?
L318 what evidence is there that GPI suppresses the formation of tumour spheres (not tumourspheres) in melanoma cells?
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