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Issues pointed by the reviewers were addressed and the 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 my concerns. I have no further comments.
The authors have addressed my concerns. I have no further comments.
The authors have addressed my concerns. I have no further comments.
Author sufficiently addressed all my comments. I have no further concerns.
I have no reservation about experimental design of this study.
I have no reservation about validity of the findings.
Both reviewers raised serious concerns that require your attention. Please address all the queries and revise the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff 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.
1. The language in the article needs revision for clarity and accuracy. For example, in the Abstract, "Upon the suppression of DHCR7, these tumor-promoting effects were counteracted, a change that coincided with the reactivation of the PI3K/AKT/mTOR signaling cascade" should be revised to: "The suppression of DHCR7 counteracted these tumor-promoting effects, which coincided with the inactivation of the PI3K/AKT/mTOR signaling pathway."
Similarly, “A wide-ranging set of methodologies was utilized to explore…” should be changed to “A wide range of methodologies was applied to explore...” Also, “DHCR7 play a key role...” should be corrected to “DHCR7 plays a key role...”
2. Figures should be cited sequentially, with individual panels labeled as A, B, C, D, etc. It is confusing for readers to find panels labeled (i), (ii). Additionally, Figure 2B is not referenced in the text.
3. Some figures lack sufficient resolution, making them difficult for readers to view clearly.
1. In the "Wound Healing Assay" section, the phrasing is unclear. Specifically, "At 300,000-500,000 cells per well, CRC cells were seeded in 6-well plates at 85-95% confluence" is ambiguous. Clarify whether cells are seeded at 85-95% confluence or if they reach that confluence after seeding.
2. The description of "Immunohistochemical staining analysis" is incorrect. The antibody labeling step should not be listed as the last step in the process.
3. In the "Subcutaneous graft tumor model" section, the inclusion and exclusion criteria for animal studies need further explanation. These criteria are not typically relevant for such studies. Additionally, the tumor volume formula (length + width + height) / 2 (mm³) seems incorrect. I suggest the authors refer to literature such as PMID: 26540189 for clarification.
4. The statistical analysis section is unclear. The term "chi-squared variances" is not standard, and further explanation is needed.
Several conclusions drawn by the authors are not supported by the data presented, and/or the validity of the data cannot be properly assessed based on the information provided.
1. The statement "DHCR7 gene expression is notably elevated in both CRC tumor samples and corresponding normal colorectal tissues, as observed in datasets from TCGA and GTEx (Figure 1A)" does not match the box plot shown in Figure 1A.
2. The authors should clarify why the concentration of 7-DHC increases in cancer tissue when DHCR7 expression rises. Typically, higher DHCR7 expression would increase sterol biosynthesis, which should lead to consumption of 7-DHC, not its accumulation.
3. The description “Figure 3D shows that DHCR7-knockdown cells expressed significantly lower levels of E-cadherin (E-Ca), N-cadherin (N-Ca), matrix metalloproteinase-9 (MMP-9), and Vimentin” does not match the immunoblots presented in Figure 3D.
4. The description of Figure 4B does not align with the image presented in the figure.
5. It is difficult to identify an increased expression of DHCR7 in Figure 5B. The bands from Vector and OE-DHCR7 seem similar.
6. The bar chart for the migration and invasion experiments in Figure 5E does not match the representative images shown in the figure.
7. The FACS image of apoptotic cells in Figure 6A shows a slight increase in the OE-DHCR7 group in HCT116 cells, which contradicts both the description and the bar chart.
8. The tumor volume growth appears too slow based on my experience, and the tumor volume in Figure 7A does not match the description in the results. The authors should review the experimental methodology and check the reliability of these findings.
This manuscript presents an in-depth investigation into the oncogenic role of 7-Dehydrocholesterol Reductase (DHCR7) in colorectal cancer (CRC). The authors employ bioinformatics analyses, in vitro functional assays, and in vivo xenograft models to demonstrate that DHCR7 overexpression promotes CRC progression via the PI3K/AKT/mTOR signaling pathway. The findings are well-supported by robust experimental methodologies, and the study addresses an important gap in understanding the intersection of cholesterol metabolism and cancer progression.
The manuscript is written in clear and professional English.
The introduction provides adequate background on cholesterol metabolism and positions DHCR7 in the context of oncogenesis.
The literature review is comprehensive and well-referenced, citing relevant studies on DHCR7 in various malignancies.
Figures are high-quality and well-labeled, and the raw data files have been made available.
Comments
The introduction could provide a clearer rationale for focusing on DHCR7 in CRC specifically. While DHCR7’s role in other cancers (breast, bladder, gastric) is acknowledged, a stronger justification for why CRC uniquely relies on DHCR7 activity would improve the framing.
Some sections contain grammatical inconsistencies, especially in Results and Discussion.
Figures could benefit from clearer legends and statistical annotations
The study is well within the scope of oncological research and cholesterol metabolism.
The research question is well-defined and novel, addressing a critical knowledge gap.
A broad range of methodologies is employed, including qRT-PCR, immunoblotting, IHC, flow cytometry, transwell assays, and xenograft models, increasing the rigor of the investigation.
Appropriate controls (siRNA knockdown, DHCR7 overexpression, normal colorectal cells) are included to validate findings.
Comments
The bioinformatics analysis from TCGA and GTEx datasets demonstrates elevated DHCR7 expression in CRC. However, it is unclear whether these results were validated across multiple independent cohorts.
The scratch wound healing assay measures cell migration, but it does not discriminate between migration and proliferation effects.
The colony formation assays support the role of DHCR7 in proliferation, but single-cell clonogenic assays would be more informative.
The study proposes that DHCR7 modulates PI3K/AKT/mTOR activation, but causality remains unclear.
Use pharmacological inhibitors to demonstrate whether DHCR7 directly regulates this pathway.
Determine whether DHCR7 knockdown affects phosphorylation levels of AKT/mTOR independent of other cellular changes.
The study provides solid experimental evidence linking DHCR7 expression to tumor progression.
Statistical analysis is appropriately conducted.
Conclusions are generally well-supported by the data.
While PI3K/AKT/mTOR involvement is suggested, direct interaction between DHCR7 and pathway components remains unclear.
co-immunoprecipitation (co-IP) or proximity ligation assays to test whether DHCR7 interacts with key PI3K/AKT/mTOR components.
DHCR7 is involved in cholesterol synthesis, yet metabolomic analysis of cholesterol derivatives (e.g., 7-DHC, oxysterols) is missing.
The study addresses a novel and clinically relevant topic.
The findings support DHCR7 as a potential therapeutic target.
Comments
Need for independent validation of bioinformatics results.
Improve mechanistic depth regarding DHCR7’s regulation of PI3K/AKT/mTOR.
Clarify whether DHCR7’s effects are sterol-dependent or independent.
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