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All remaining concerns of the reviewers were adequately addressed, and the revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
The authors have addressed all the comments.
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Please address remaining issues pointed by the reviewer #3 and amend manuscript accordingly.
The author has not sufficiently addressed all my questions and concerns. While I understand the limitations and experimental constraints of the laboratory, these should not be used as a justification to omit essential experiments, such as Western blot analysis. In its current form, I believe the manuscript is not suitable for publication.
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Thank you for the response. As stated by the authors in their responses to various comments, the inclusion of additional cell lines was not recommended by the reviewers for any of the studies. However, minor wet lab experiments utilizing the same BHT101 cell lines were suggested. These experiments are essential to enhance the manuscript's quality and are therefore mandatory. Some of the comments to authors' responses to previous comments are addressed below:
1. In response to comment 1:
The authors mentioned that they addressed the question in the manuscript between lines 350–365. However, this section does not sufficiently explain the issue raised and is unsatisfactory. A more detailed explanation is required.
2. In response to comment 2:
Performing a western blot experiment is a fundamental approach to demonstrate protein expression. It is crucial to include such an experiment in this paper, at least for two of the proteins mentioned in the manuscript.
3. In response to comment 3:
The authors have not provided a specific or detailed response to the question, as the explanation in the referenced lines is inadequate. It is recommended to propose possible pathways, supported by relevant references. Additionally, the authors should investigate whether SK4 translocates into the nucleus under the conditions described in the study, using immunostaining techniques.
4. In response to comment 5:
If there is insufficient data to support the role of SK4 in invasion or proliferation, the corresponding section should be removed from the manuscript. The inclusion of additional cell lines is not needed for these studies. This aspect should be mentioned only in the discussion as a potential hypothesis.
5. In response to comment 6:
If the authors lack strong data to demonstrate immune infiltration, all statements referring to this should be removed from the manuscript. This aspect should be mentioned only in the discussion as a potential hypothesis.
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Please address concerns of all reviewers and amend manuscript accordingly
Overview
In this study, the author identified SK4 as one of the genes associated with Thyroid Carcinoma progression using TCGA and TIMER database data. The author then validates SK4 functions by overexpressing the SK4 gene in the BHT101 cell line. The author showed that, while SK4 overexpression does not change global gene expression patterns, it results in significant changes in alternative splice expression of multiple genes.
Overall, I think the manuscript is well-written and easy to follow. The figures are quite well represented, though some labels are still missing. The data is interesting since it is surprising that potassium channels could affect DNA integrity and splice function.
My only criticism of this manuscript is the lack of cell biology validation of alternative splice. Specifically, I wonder whether these alternate spliced genes result in protein dysfunction. While it is easy for me to ask that question, I am aware that all of the genes identified in this study are poorly understood. Apart from that, I have a list of a few concerns below.
I would recommend a minor revision before publication.
Major Concern
1. Page 13, lines 180 – 182: While the evidence is convincing that KCCN4 is associated with Thyroid Carcinoma, to claim that SK4 'promotes' cancer progression is an overclaim. I would suggest rewriting it to 'SK4 expression is highly associated with THCA progression', or something of that nature.
2. Figure 2: For the overexpression experiment, I would like to see that overexpression is also successful at the protein level. For example, the author should run Western Blot or Immunofluorescence. I prefer Immunofluorescence since overexpressed-SK4 localization could also suggest that the overexpressed protein is functioning.
3. Figure 2C: I could not find any details on how the 'Cell Invasion' experiment was performed. Also, could the author describe the bright green area in the lower panel as well?
4. Figure 4: Was the statistical comparison in the RT-qPCR experiment done to OE vs NC as a group or as an individual sample? I find that the expression difference as measured by qPCR is not very convincing; however, I still think that the author's claim up to this point is still valid. I would consider moving these panels to supplementary data instead of the main figure.
5. Figure 5: Since most AS-dysregulated genes are related to DNA damage, I suggest the author perform a cell-based experiment to show that SK4-OE induces more DNA damage. A few simple experiments, such as gH2AX fluorescence staining or Annexin V labeling, come to mind. A few experiments like these would strengthen the author's claim.
Minor Concern
1. Page 7, line 32: What is 'AS' pattern here, Alternative Splicing?
2. Figure 1: KCCN4 should be changed to SK4 to match the rest of the text.
3. Figure 2A: There should be an x-axis label, not just a tick label.
4. Figure 3D and Figure 6A: What should the color bar value indicate? It seems like the color bar label is missing.
Please see comments above.
All findings and claims are reasonable.
This study provides insights into the role of the potassium channel protein SK4 in papillary thyroid cancer (PTC) progression, particularly focusing on its unexpected influence on alternative splicing. The authors demonstrate that SK4 overexpression in BHT101 thyroid cancer cells enhances proliferation and invasion, consistent with previous findings in other cancer types. However, the study's most significant contribution lies in its comprehensive transcriptomic analysis, which reveals that SK4 predominantly modulates gene expression through alternative splicing rather than transcriptional regulation. However, the author should improve the manuscript:
1. Tighten the writing throughout - remove redundancies and focus on key points.
2.Improve figure quality and ensure all are properly labeled with statistical information.
3.Carefully proofread for grammar and language issues throughout.
1. Author should explain their choice of BHT101 cells. As a thyroid carcinoma cell line, why its expression of SK4 is so low? If the SK4 expression is higher in BHT 101 cells, how about using knockdown method to study its function?
2. Author should use other method to prove the overexpression SK4 protein like western blot.
3. More information needed on bioinformatics tools and parameters used.
1. Author should validate key findings in additional cell lines or patient samples.
2. Author should strengthen the mechanistic link between SK4 and splicing regulation-consider additional experiment to elucidate this.
The study investigates the impact of SK4 overexpression on thyroid cancer using BHT101 cell lines. While the study has a robust bioinformatics foundation, the molecular biology aspects require further emphasis to substantiate the claims made in the manuscript. Here are the key revisions needed:
1. Despite a 50-fold overexpression of the SK4 gene in the cell lines, the RT-PCR data shows minimal differences in gene expression across nearly all genes studied, including those related to alternative splicing. Please explain this. It is crucial to provide evidence that these pathways exert significant effects on the cells. Given that the title emphasizes the alternative splicing aspect of the study, please provide more extensive data to substantiate this focus.
2. Please analyze the expression of protein levels that are indicated to undergo alternative splicing corresponding to SK4 overexpression. It will be interesting to see if the AS is affecting the protein expression levels or produce mutated/truncated protein.
3. Suggest potential pathways through which SK4, a membrane-bound protein, might influence the alternative splicing of specific genes in the discussion. Since you have emphasized on the AS part of the study as a novel finding, please provide more data and interpretation to support your finding, especially since the mRNA expression levels are not hugely altered in the RTPCR data in the SK4 overexpressed cells as mentioned in comment 1.
4. Please include detailed information about the patient study, including ethical approval, in the Materials and Methods section. Also include a detailed explanation of patient categories, including the basis for grouping, inclusion, and exclusion criteria, in the Methods section. Please clarify what is meant by “normal samples.” Are these healthy controls matched for age and gender?
5. Invasion and Proliferation assay: The method for the invasion study is not described in the Methods section. Clarify whether the CCK8 assay was used for both proliferation and invasion studies. If so, specify this in the Methods section. The CCK8 assay alone may not be sufficient to demonstrate cell proliferation and invasion. Were you able to identify any specific gene expression changes related to proliferation and invasion in the RNA-seq data and if so, validate those findings with RTPCR/WB and correlate these with the assay results. Additionally, consider using flow cytometric analysis of cell cycle stages to provide a more comprehensive assessment of proliferation, rather than relying solely on CCK8 assays. Please report whether cell numbers were counted prior to treatment.
6. Immune Infiltration Validation: Line 168: Indicate whether any wet lab experiments were conducted to validate the immune infiltration results. Correlate TIMER-based findings with any cell culture or patient sample studies. Were you able to find any genes that are responsible for immune infiltration in the RNAseq data?
7. The manuscript requires revision to use appropriate scientific terminology and phrasing.
8. Expand gene names when first mentioned to ensure clarity.
The study has focused more on the bioinformatics foundation, the molecular biology aspects require further emphasis to substantiate the claims made in the manuscript.
Please provide more molecular biology data to validate the findings.
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