Regulation of TREM1-Mediated Inflammation in Hepatocellular Carcinoma Cells
Round 1
Reviewer 1 Report
Comments to Author
The authors describe the effect of vitamin D on TREM-1 expression in HCC cell line (HepG2) cells and in epithelial cells transformed with SV40 large T antigen (THLE-2) as a control. They showed that vitamin D significantly attenuates the expression of mediators of inflammation and thus, might be used as a therapeutic drug. The content of the manuscript is attractive in terms of future innovation in cancer treatment.
Major point:
- The authors performed the immunofluorescence assays to examine the protein expression levels of target gene though out the manuscript (from Fig.1 to Fig. 6), however, the assay can provide only a semi-quantitative, and western blot analysis should be done for quantification analysis of protein expression.
- In Fig.5, the authors examined the expression levels of NF-kB gene, however, the activation of NF-kB signaling pathway should be evaluated by the phosphorylation levels of NF-kB signaling molecules such as IKK, protein level of IkBα, or processing of p100.
- In Fig. 7, please provide the quantification data for the Scratch test to clearly show the statistical difference.
Minor points:
- Abstract; Please describe clearly new findings provided by this study.
Author Response
Comment: The authors describe the effect of vitamin D on TREM-1 expression in HCC cell line (HepG2) cells and in epithelial cells transformed with SV40 large T antigen (THLE-2) as a control. They showed that vitamin D significantly attenuates the expression of mediators of inflammation and thus, might be used as a therapeutic drug. The content of the manuscript is attractive in terms of future innovation in cancer treatment.
Response: Thank you for your positive comments.
Major point:
Concern 1: The authors performed the immunofluorescence assays to examine the protein expression levels of target gene though out the manuscript (from Fig.1 to Fig. 6), however, the assay can provide only a semi-quantitative, and western blot analysis should be done for quantification analysis of protein expression.
Response: Thank you for your comment and suggestion. We have done western blot and the results have been included in the revised manuscript.
Concern 2: In Fig.5, the authors examined the expression levels of NF-kB gene, however, the activation of NF-kB signaling pathway should be evaluated by the phosphorylation levels of NF-kB signaling molecules such as IKK, protein level of IkBα, or processing of p100.
Response: Thank you for your suggestion. We have included the immunofluorescence and mRNA expression studies for IκB kinase (IKK) in HepG2 cells as well as SNU387 cells (HCC cell line).
Concern 3: In Fig. 7, please provide the quantification data for the Scratch test to clearly show the statistical difference.
Response: Thank you for your suggestion. We have analyzed the percent area not having cells (less than 10 cells in the ROI were considered normal as at 0 hr control there were some cells in the scratch area even after washing with PBS). The results have been included in the revised manuscript along with the scratch test figure.
Minor points
Concern 1: Abstract; Please describe clearly new findings provided by this study.
Response: Thank you for your suggestion. We have modified the abstract and have included the main results in the abstract section.
Reviewer 2 Report
The paper presents an approach to hepatocellular carcinoma from the perspective of specific inflammatory markers. The study considered therapy aimed at changing the values ​​of the TREM-1 (triggering receptor expressed on myeloid cell) marker. At the same time, a series of inflammatory markers that predispose the liver to carcinogenesis were analyzed. The presentation made by the authors in the article is very detailed. They propose an improvement in conventional treatment that would limit the progression of HCC.
Although the authors' approach is very detailed, I have some comments that could improve the quality of the presentation of the results.
1. In section 2. Results I recommend presenting only personal results. The comparative presentation or presentation of the data from the specialized literature is reported in the Discussions section.
2. Before the concrete presentation of the study results (2. Results) I recommend the introduction of section 4. Material and methods.
Also this section (Material and Methods) should be more concise. Other information is required in this section, e.g. number of samples analyzed, etc. This is now presented only in the legend of the figure, but it is not enough.
3. The presentation of mean values ​​and standard deviations should be revised in view of the small number of values ​​analyzed. Perhaps the median values ​​together with the quartiles or the minimum and maximum values ​​would be more useful.
4. The Conclusions section needs to be redone. In this section the authors make numerous references to the literature, which is not necessary. The conclusions must be clear, concrete, and based in particular on the study conducted by the authors.
Author Response
Comment: The paper presents an approach to hepatocellular carcinoma from the perspective of specific inflammatory markers. The study considered therapy aimed at changing the values ​​of the TREM-1 (triggering receptor expressed on myeloid cell) marker. At the same time, a series of inflammatory markers that predispose the liver to carcinogenesis were analyzed. The presentation made by the authors in the article is very detailed. They propose an improvement in conventional treatment that would limit the progression of HCC. Although the authors' approach is very detailed, I have some comments that could improve the quality of the presentation of the results.
Response: Thank you for your comments. We have revised the manuscript as per the suggestions.
Concern 1: In section 2. Results I recommend presenting only personal results. The comparative presentation or presentation of the data from the specialized literature is reported in the Discussions section.
Response: Thank you for your suggestion. We have modified the Results section as per the suggestion by the reviewer in the revised manuscript.
Concern 2: Before the concrete presentation of the study results (2. Results) I recommend the introduction of section 4. Material and methods. Also this section (Material and Methods) should be more concise. Other information is required in this section, e.g. number of samples analyzed, etc. This is now presented only in the legend of the figure, but it is not enough.
Response: Thank you for your comments and suggestion. The Reports journal suggests formatting the manuscript in order of Introduction, Results, Discussion followed by Material and Methods (https://www.mdpi.com/journal/reports/instructions#preparation), so we have followed these instructions. The Material and Methods section has been modified in the revised manuscript.
Concern 3. The presentation of mean values ​​and standard deviations should be revised in view of the small number of values ​​analyzed. Perhaps the median values ​​together with the quartiles or the minimum and maximum values ​​would be more useful.
Response: Thank you for your comment and suggestion. We have presented the data as mean ± SD because the median is more reliable when outliers are present in the data. We did not have outliers in the data, so we chose to used mean. As per the suggestion by the reviewer, we have included the minimum and maximum values in the revised manuscript as Supplementary Table 1.
Concern 4: The Conclusions section needs to be redone. In this section the authors make numerous references to the literature, which is not necessary. The conclusions must be clear, concrete, and based in particular on the study conducted by the authors.
Response: Thank you for your suggestion. We have revised the Conclusion section as suggested by the reviewer.
Round 2
Reviewer 2 Report
The authors followed the recommendations made.
Section 2. Results and 4. Material and methods have been significantly improved.
However, I still consider that section 4. Material and methods must be moved before section 2. Results.
Although the authors claim that they followed the recommendations for formatting the manuscript (https://www.mdpi.com/journal/reports/instructions#preparation), I analyzed all those recommendations and this does not appear.
In fact, it is totally inappropriate for the protocol and the method of analysis (4. Material and methods) to be present after discussions.
For results presented in section 2. Results the authors provided additional material. The additional information now provides a complete picture of the analyzed parameters.
The 5. Conclusions section has been revised and the authors now present a clear form of them.
I consider that the article can be published in this form, but I maintain my observation regarding the position of section 4 (4. Material and methods)
