The Role of miRNA-221 and miRNA-34a in Non-Melanoma Skin Cancer of the Head and Neck Region

Round 1

Reviewer 1 Report

Why you have selected these miRNAs for the research work? Discussion required more focus and grip over the subject

Author Response

R1 - Why you have selected these miRNAs for the research work? Discussion required more focus and grip over the subject

 

Dear reviewer, thank you for the quick response and for your interest in our work. We wanted to choose an oncogene miRNA and a tumor suppressor miRNA for this study. Among all miRNAs, we have chosen these two for different reasons, which I will further detail.

A lot of human tumor tissues, including those from liver cancer, breast cancer, prostate cancer, colorectal cancer and acute myeloid leukemia have been found to overexpress miRNA-221. Also, poor prognosis factors such as metastasis, lymph node capsular infiltration and advanced tumor stage are generally associated with high levels of miRNA-221 expression. Melanoma, which has a very poor overall prognosis, expresses high levels of miRNA-221. We found these data very challenging and we thought that it could also have a role in non-melanoma skin cancer, which has an increase incidence among cancer sites, despite its improved prognosis. The NMSC expressed high levels of miRNA-221, confirming our hypothesis. The high levels of miRNA were also associated to the positive surgical margins, which may play a role for the future tumor excision process.

As a tumor suppressor, miRNA-34a can control the cell cycle and prevent tumor cells from migrating, spreading and invading the healthy tissue. Also, a few studies concluded that irreversible inactivation of miRNA-34a may be another defensive mechanism of the tumor cells. The arising and growth of numerous malignant tumors, such as lung adenocarcinomas, breast cancer and esophageal cancer are strongly correlated with miRNA-34a. The role of miRNA-34a in the development of non-melanoma skin cancer has been described in a few studies, mostly in blood samples and for patients with BCC, not all NMSC, and it is generally associated with poor prognosis factors. We discovered low levels of miRNA-34a directly in the tumor tissue in both BCC and SCC, but unfortunately, the data is not statistically significant due to the small sample of cases, but it can lead to future research regarding the role of this miRNA in SCC and overall survival.

The frailty of our study was the small sample size, due to the necessity of dividing the probes into tumor samples for BCC, BSC and SCC, but the research has also has some strong points, such as the possible role of miRNA-221 in tumor microinvasion and the downregulation of miRNA-34a in SCC, which is not very debated in the literature. There are also authors who say that miRNA-34-a is not downregulated in NMSC, but we demonstrate the opposite. Further, I attach the article links.

https://www.frontiersin.org/articles/10.3389/fcell.2021.640587/full

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459507/

https://www.hindawi.com/journals/jo/2019/7252013/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8891816/

Reviewer 2 Report

The authors present the role of miRNA221 and miRNA34a in the tumor progression of Non-melanoma skin cancer. The authors have given a decent introduction but the overall study is not very sound and the conclusions drawn are based on a literature survey and not actual experimental evidence. In my opinion, a lot of work is needed to justify the hypothesis and role of these miRNAs in NMSC progression. The cohort size is small, as mentioned by the authors, and may not represent the true miRNA levels.

Apart from this, I am also not very convinced by the qPCR data in fig. 3. The difference between the expression level of miRNA34a is not stark and does not seem significant. Similarly, the difference in the expression level of miRNA221 in BSC is not clear as to whether there is an increase or decrease in the tumor when compared to adjacent tissue, which is very variable between the two samples. The authors could have explained it more deeply.

The discussion was extremely long. I felt like it is a repetition of the introduction. Several paragraphs could be concised into one. Also, there is no possible explanation for the overexpression of miRNA221 in BCC. The authors simply stated that they could not find anything in the literature.

Experimental evidence is clearly lacking. Adding a few experiments, like ROC curve or overexpression of these miRNA in melanoma cell lines to prove their roles in tumor progression would definitely support t he data and. make this study more meaningful.

Author Response

R2- The authors present the role of miRNA221 and miRNA34a in the tumor progression of Non-melanoma skin cancer. The authors have given a decent introduction but the overall study is not very sound and the conclusions drawn are based on a literature survey and not actual experimental evidence. In my opinion, a lot of work is needed to justify the hypothesis and role of these miRNAs in NMSC progression. The cohort size is small, as mentioned by the authors, and may not represent the true miRNA levels.

Dear reviewer, thank you for the quick response and for your interest in our work. We are really sorry for leaving the impression that our conclusions were drawn from the literature and not from our results. We wanted to choose an oncogene miRNA and a tumor suppressor miRNA for this study. As we mentioned, our 38 patients cohort had to be divided into different hystological subtypes due to the variety of NMSC subtypes but we intend to continue this research and extend the number of patients.

Apart from this, I am also not very convinced by the qPCR data in fig. 3. The difference between the expression level of miRNA34a is not stark and does not seem significant. Similarly, the difference in the expression level of miRNA221 in BSC is not clear as to whether there is an increase or decrease in the tumor when compared to adjacent tissue, which is very variable between the two samples. The authors could have explained it more deeply.

The figure 3 presents the results very briefly and we already mentioned that the result of the miRNA34-a downregulation in NMSC it is not statistically significant. To be more specifically, we have revised the miRNA expression level results. Compared to the adjacent tissue, miRNA-34a shows an under-expression without statistical significance in all types of NMSC. The tumors were shown to express higher levels of miRNA-221 than adjacent tissue only in the SCC cases with statistically significance. Regarding the BSC, we had only two cases due to the rarity of this hystological type ,so we could not formulate any conclusion. If you consider, we can remove it from the study.

The discussion was extremely long. I felt like it is a repetition of the introduction. Several paragraphs could be concised into one. Also, there is no possible explanation for the overexpression of miRNA221 in BCC. The authors simply stated that they could not find anything in the literature.

Thank you for this remark. The first variant of the article had way shorter chapter of discussions, but it was returned from the editor because the minimum required number of words is 4000. For this reason, a lot of information was added in introduction and discussion chapter. By removing it, we will be taken back to the initial report.

Experimental evidence is clearly lacking. Adding a few experiments, like ROC curve or overexpression of these miRNA in melanoma cell lines to prove their roles in tumor progression would definitely support t he data and. make this study more meaningful.

We sincerely appreciate your idea and we will take it into consideration, because we want to extend our research. Unfortunatelly, we cannot do it in 10 days as the journal requires specifically.

Reviewer 3 Report

 The role of miRNA-221 and miRNA-34a in non-melanoma skin 2 cancer of the head and neck region

 

 

 

A brief summary

This study aimed to determine the function of miRNA-221 and miRNA-34a in non-melanoma skin cancer of the head and neck region, using quantitative Real-Time RT-PCR. The authors describe overexpression of miRNA-221 in different subtypes of NMSC compared to normal tissues. In addition, they describe downregulation of miRNA-34a in different subtypes of NMSC compared to normal tissues.  

 

General concept comment :

The major points that should be adresse are :

Scientific writing on genetic topics is weak, especially in the introduction section, and these materials should be rephrased.

 

Specific comments (might be redundant with the general comments for some points.

1. Statistical parameters must be mentioned in the abstract where you have presented the results

2. Considering that in the materials and methods section, 3 out of 41 people were excluded from the study, it is better to report 38 people here instead of 41 people.

3. In the abstract part, you have not mentioned any of the materials and methods and it should be mentioned.

4. There is no need to include Figure 1 in the introduction section because it does not help to better understand the existing version and it is suggested to be removed.

5. The type of referencing in the entire text is not appropriate, for example, at the end of the first paragraph, 1 to 13 are introduced, or in the second paragraph, 14 to 21 are introduced.

6. Lines 51 and 52 should be rephrase scientifically.

7. In the introduction, there is no need to give the details of the biosynthesis process of micro RNAs. In addition, there is no proper writing of these parts from the scientific point of view.

8. There is no need to include Figure 2 in the materials and methods section because it does not help to better understand the method and it is suggested to be removed.

9. In line 154, it is better to write quantitative real time RT-PCR

10. Rephrase lines 166 to 168 about the PCR program because the extension step has not been implemented.

11. What is the reason for the sequence of miRNAs in Table 1?

12. Correlation of the contents in the discussion section is not appropriate and there is a scattering of scientific writing and it is better to revise it.

Author Response

R3- Specific comments (might be redundant with the general comments for some points).

1. Statistical parameters must be mentioned in the abstract where you have presented the results

Dear reviewer, thank you for the quick response and for your interest in our work. The statistical parameters were added in the abstract.

2. Considering that in the materials and methods section, 3 out of 41 people were excluded from the study, it is better to report 38 people here instead of 41 people.

We have corrected the total number of cases to 38.

3. In the abstract part, you have not mentioned any of the materials and methods and it should be mentioned.

The materials and methods have been also added to the asbtract now.

4. There is no need to include Figure 1 in the introduction section because it does not help to better understand the existing version and it is suggested to be removed.

Thank you for this remark. We wanted to show a clinical part of this research. As you suggest, the figure 1 was removed

5. The type of referencing in the entire text is not appropriate, for example, at the end of the first paragraph, 1 to 13 are introduced, or in the second paragraph, 14 to 21 are introduced.

Thank you for the remark. We know that it is not the best way to type the referencing, but we chose this style because the text contains a lot of numbers and parenthesis, so we found it a litle bit to hard to follow. If it is possible, we would like to keep it this way.

6. Lines 51 and 52 should be rephrase scientifically.

These lines were modified.

7. In the introduction, there is no need to give the details of the biosynthesis process of micro RNAs. In addition, there is no proper writing of these parts from the scientific point of view.

We removed this part as you suggested.

8. There is no need to include Figure 2 in the materials and methods section because it does not help to better understand the method and it is suggested to be removed.

The figure was removed.

9. In line 154, it is better to write quantitative real time RT-PCR

The line is now modified.

10. Rephrase lines 166 to 168 about the PCR program because the extension step has not been implemented.

The lines were rephrased.

11. What is the reason for the sequence of miRNAs in Table 1?

There is no reason for the sequence. We only present the miRNA assay primers sequences used in the study. If you consider it unnecessary, we can remove it.

12. Correlation of the contents in the discussion section is not appropriate and there is a scattering of scientific writing and it is better to revise it.

Thank you for this remark. The first variant of the article hada shorter chapter of discussion and it was correlated only to the results, but it was returned from the editor because the minimum number of words required is 4000. For this reason, a lot of information was added in introduction and discussion chapter. By removing it, the article will be returned again, as the initial form.

Round 2

Reviewer 2 Report

The authors have revised the manuscript to the best of their ability. They repeated the qPCR analysis and again found the downregulation of miRNA34a but statistically insignificant. However, my concern in the first point was that if they increase the cohort size and find that miRNA34a levels remain unchanged from the adjacent tissue, then the whole hypothesis falls apart. So, in absence of a larger cohort, it is not advisable to call it a downregulation if it is statistically insignificant. Also, I would advise authors to change/frame the discussion in a more scientific way rather than repeating the introduction to cover 4000 words. They could ask for grace time to perform the experiments required to strengthen the study, like overexpression analysis or ROC curve.

Author Response

Dear Reviewer, thanks again for the quick response. We appreciate your recommendations and would clearly like to expand our research. Unfortunately, I will not be able to do it as soon as you suggest it because we have no more financial resources. The research was conducted and funded by the Doctoral School of the University of Medicine and Pharmacy. I am not able to get any other funding through this program and getting another grant will take a long time. I've changed the term from "downregulated" to "altered expression" if you think it's appropriate. Thank you very much for all your advice.

 

Kind regards,

Tiberiu Tamas

Reviewer 3 Report

 

The authors have addressed many of the requested comments, but there are still some that remain, which are listed below

 

1. The type of referencing in the entire text is not appropriate, for example, at the end of the first paragraph, 1 to 13 are introduced, or in the second paragraph, 14 to 21 are introduced.

2. In the method section, lines 167 to 169, according to the authors, the total volume of the reaction is 10 microliters, but their total is more than 10 microliters, and the unit of volume is not mentioned for the master mix.

3. The real time RT-PCR program is not mentioned in the materials and methods section

4. What is the reason for the sequence of miRNAs in Table 1?

5. Correlation of the contents in the discussion section is not appropriate and there is a scattering of scientific writing and it is better to revise it.

 

Author Response

1. The type of referencing in the entire text is not appropriate, for example, at the end of the first paragraph, 1 to 13 are introduced, or in the second paragraph, 14 to 21 are introduced.

The referencing method was changed as you suggested 

2. In the method section, lines 167 to 169, according to the authors, the total volume of the reaction is 10 microliters, but their total is more than 10 microliters, and the unit of volume is not mentioned for the master mix.

Thank you! We have corrected the mistake

3. The real time RT-PCR program is not mentioned in the materials and methods section

The program is now introduced.

4. What is the reason for the sequence of miRNAs in Table 1?

As we explained before, there is no reason for it. We have removed it from the table.

5. Correlation of the contents in the discussion section is not appropriate and there is a scattering of scientific writing and it is better to revise it.

We have revised it the last time and now again and I’m afraid that we have no clue how could we improve it. If you have any other recommendations, please let us know. Thank you again for your quick response and for you guidance.