Exploring miRNA Biomarkers in Major Depressive Disorder: A Molecular Medicine Perspective

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

MDPI

 

Current issues in molecular biology

 

Manuscript: MDPI-cimb-3201648

 

The authors studied exploring miRNA biomarkers in major depressive disorder: a molecular medicine perspective. The authors presented classification of ribonulcleic acids (Fig. 1), overview of the trajectory (Fig. 2), demographic characteristics of pattern group versus control group (Table 1), disregulations of specific miRNAs in major depressive disorder (Fig. 3), application of independent t-test (Table 3), ROC analysis (Fig. 4), area under the receiver operating curve for biomarkers (Table 4).

 

The authors concluded and reported that this research may pave the way for new avenues in molecular medicine, biochemistry, neuropsychiatry, and neuropsychopharmacology, by identifying molecular trigger and more specific markers. The authors have provided novel information in major depressive disorder.

 

 

 

Author Response

Dear reviewers, 

Thank you for pointing out our shortcomings! We have worked on revising our manuscript, and, as required, we have made the following changes: 

- Abstract: re-wrote it to better summarize the whole manuscript - intoduction, aim, methods, results and discussions. 

- Introduction: we considered trimming it to make it more precise and on point. First we introduced the concept of MDD, then we introduced RNAs and miRNAs, and then we wrote about the potential connection between miRNA dysregulation and MDD while stating the aim of the study. 

- Materials and Methods: we presented this section in a more clear and precise way - study design and population - miRNA manipulation - statistical analysis. We tried to explain the methodology better than the previous manuscript. 

- Results: we made major changes in this section. We structured it differently, in 3 subsections, each with a clear purpose: short summary of the study population - t-test results - ROC curve results. In order to prove data accuracy, tables were provided across all subsections. 

- Discussions: significant changes have been made in this section too. We discussed our results, specifically the miRNAs that showed significant dysregulation in our study, and how our results correlate with other studies. We added two paragraphs about the limitations of our study. 

- Conclusions: we believe that this section is now more concise and better summarizes our findings. 

Overall, we made significant changes to the language we used, in order to comply to academic writing standards. We proof-read the manuscript twice in order to remove any typographic errors. 

While this version of the manuscript may still need revision, we hope that this is an improvement. 

In the name of the authoring team, and as the submitting author, I would like to present our gratitude for helping us turn this manuscript into a good piece of medical literature! Thank you! 

Kind regards, 

Norberth Varga

Reviewer 2 Report

Comments and Suggestions for Authors

The purpose of this comparative study is to identify specific miRNA biomarkers associated with Major Depressive Disorder (MDD) in order to potentially improve the diagnosis and disease management by enhancing the existing diagnostic panel.

As part of this case-control study, miRNA expression analysis was performed in 10 MDD patient samples and in a control group consisting of eight healthy individuals. miRNA expression was analyzed using real-time PCR.

Considering the numerous knowledge gaps that still exist and the requirement for trustworthy biomarkers for early MDD diagnosis and disease management, this study is relevant and essential.

 The manuscript is written in average quality English that needs improvement and partially conforms to an academic writing style. The majority of the referenced works are recent and relevant publications, but the manuscript also includes irrelevant and trivial information that unnecessarily lengthens the text and does not focus on the main research topic.

 Overall, the experimental approach employed is suitable for testing the hypothesis proposed by the authors. The study design is adequate, but it is inadequately represented in the text of the article, as evidenced by the poor description of methods and the confusing interpretation of results.

 

Title and Abstract

 The title of this article is well-written and accurately conveys its goal and content.

 The abstract section is not a complete and objective representation of the article and does not include all relevant information. The main results described in the abstract do not accurately reflect the results of the study, in addition, information on the number of patients included in the study in the groups is also not included.

 The selected keywords are relevant to the topic of this study.

 Introduction

 The introduction needs to be substantially improved and is currently unfinished. It lacks a clearly stated primary aim of the study and includes information that is somewhat out of date and unrelated to the study's particular issue.

 Particular points that need to be addressed:

 -       In lines 52-54, the wording of the sentence is not clear to the reader and needs to be improved.

-       To improve the readability of the text, instead of using the full name of the disease, it is recommended to use the abbreviated version (an acronym "MDD") throughout the manuscript, which is indicated on line 52 of the text. This recommendation also applies to other abbreviations that are indicated and referred to in the text below, such as cRNA, miRNA, and others.

-       Regarding the introduction of the manuscript, the first paragraph of the subsection "1.2 Biogenesis of miRNA and Their Classification" in lines 67-79 includes insignificant and trivial information about RNA classification, which does not seem relevant to the reader in the context of this article. If the article focuses on miRNA, then the introduction should contain relevant information about it, without devoting an entire subsection to the description of RNA classification, which is also supplemented by a redundant and uninformative image 1. it would be advisable to completely remove this subsection from the article and integrate some of the most important facts from it into the beginning of the next paragraph part.

-       In lines 71 and 72 an incorrect abbreviation for noncoding RNA is used. The abbreviation "NCNAs" is used, which stands for nanocarriers based on nucleic acids. In line 74, after the correct abbreviation "ncRNAs", another abbreviation "(NCNAs)" is given in brackets, the meaning of which is not clear.

-       Addressing Figure 1, the information displayed has little added value and is not informative. This information in the main text already seems redundant, because it is basic information about this topic. The image design also does not meet the requirements. For example, in the text box on the left of the image, the term "microRNA" is split in two lines without using a hyphen, and the description of the image does not include explanations of all the abbreviations used in the image. I would recommend that this image be removed from the manuscript.

-       Since the study was conducted using human samples, this sentence in lines 85-86 is redundant and general. In addition, Latin terms need to be in italics.

-       The paragraph in lines 101-104 contains irrelevant information. I would recommend removing it, as it unnecessarily lengthens the text.

-       The abbreviation in the line 123 "HPA" is not explained.

-       Line 125 has redundant text in square brackets, possibly an unformatted reference.

 Methods

 The methods section contains all the necessary subsections that describe the research design, sampling and patient inclusion criteria and sample preparation. Information of various data analysis methods and software used for the analysis is also provided.

In general, the manuscript's results are partially reproducible based on the details given in the methods, considering it doesn't contain much information.

 Line 149 mentions ROC curve analysis, but gives the reader insufficient information about this statistical method. When mentioning this method for the first time, it is recommended to mention its full name. In addition, it would be necessary to clarify the main principle of the method (this applies to the results section) - for what purpose this method is used, represented by AUC and other related values ​​and other necessary information that would allow the reader to better understand the obtained results.

 Results

 The results section has its own shortcomings, which are described specifically point by point:

 -       The sentence in lines 162-164 needs improvement for clarity.

-       In line 167, there is no need to bracket specific miRNAs because "etc." indicates that they are not the only miRNAs, thereby unnecessarily lengthening the text, because the mentioned biomarkers are not specific - not all 15 are mentioned.

-       The subsection "3.3 Statistical Analysis" included in the results section is very confusing for the reader - the procedure for renaming the samples is not clearly understood. Lines 209-212 indicate how nine samples (BM7-BM15) are renamed, but this is unusual given the fact that the beginning of the results section states that fifteen hsa-miRNAs show significant variations in fold regulation, suggesting that these 15 potential biomarkers will also be included in further statistical analyses. No criteria are given as to why exactly these nine miRNAs are renamed to alternative names.

-       It is also not clear why these alternative names have not been introduced at the very beginning of the results part, especially in Table 2, where the alternative names of miRNAs could be present instead of the "Number" column or in a completely new column, and the full names should not be included in the following text in order not to unnecessarily lengthen and complicate text for the reader. Lines 235-236 reference that samples BM1-BM15 are listed in Table 2, which (1) Indicates that all 15 miRNAs are named in the alternative names, not just nine; (2) The reader is led to believe that Table 2 should contain all 15 miRNAs with their abbreviated names, but this is not the case. Furthermore, the abbreviations are given in a mixed order and it is not even clear to the reader which miRNA ID corresponds to the BM1, BM2 and BM6 mentioned in the results in line 237.

-       It should be noted that Figure 3 would also be easier for the reader to understand if miRNA ID names were replaced by the abbreviated names deciphered in Table 2, or both names were included.

-       In reference to the research hypothesis presented in lines 213–216, why were the particular biomarkers "BM1, BM2, BM3, and BM15" chosen for testing, and which markers fall under the "and others" category? This hypothesis needs to be changed because it is imprecise and unclear regarding its development.

-       The miRNA intervals in lines 236-237 are confusing. Why, for example, instead of "BM7, BM8 to BM15" is it not written "BM7 to BM15"?

-       The description of Table 3 does not provide explanations of the abbreviations used in the table, for example "df". It should also be mentioned that the presentation of p-values ​​differs from that used in Table 2 and Figure 3, so it is recommended to present these values ​​in a uniform style. Another recommendation is to highlight those p-values ​​in bold , which is below the 0.05 threshold. In addition, all values presented in the table ​​should have an equal decimal places.

-       The expression "Analysis starts with biomarker hsa-miR-532-5p" in line 242 is unusual - information is not being presented in an academic manner. In addition, in the following text in lines 243-244 it is indicated that the result for the particular miRNA demonstrates statistical significance in its differential expression between case and control samples, but no p values ​​or any actual statistical value is indicated to confirm this statement.

-       Figure 4 has a number of design issues: (1) the axes' numerical values are too small; (2) a graph legend would need to be added for clarity; (3) the biomarkers' names are arranged inconsistently across all graphs, with their placement above the y-axis' numerical values; and (4) the image description fails to explain what BM7-BM15 stands for, which is essential information for readers to understand the image without having to read the publication's text.

-       The results described in lines 263-265 lack theoretical justification. The reader should be informed about what this index represents and what its values ​​indicate in the context of the results obtained.

 Discussion and Conclusions

 The discussion part is weak and has a number of issues. The opening section of the discussion lacks a general summary of what was done as part of this study and what was found as a result.

 Only a small part of the results is discussed - mainly two miRNAs in detail (miR-532-5p and hsa-miR-339-5p) and information about other miRNAs examined in the study is missing. There is also no mention of hsa-miR-136-3p that showed statistically significant downregulation in this study and its potential reasons. Thus, the discussion section needs to be expanded. Because the results section stated that the authors' hypothesis was being tested, a summary of whether this hypothesis was confirmed or partially confirmed is missing. Another important part missing from this discussion is a critical approach to the data obtained, the methods used, the statistical power, the study design and other conditions that could possibly affect the results of the study.

 

The discussion leaves an impression that the authors believe that the work has no limitations, and the obtained results are sufficiently convincing to be implemented in real medical practice, but it is not discussed whether these results do not require additional validation in a larger sample set. Especially because the authors themselves state in line 182-183 that the obtained results are in contrast to another study. This fact is also not explained in the discussion part.

 In line 281 – it might not be clear to the reader of which top three authors are talking about, this statement needs to be expanded and specified.

 The conclusion part is incomplete. Conclusions must be specific and it is not advisable to indicate the names of the miRNAs examined in the study only partially (four and the others are marked with "etc"), moreover, the conclusion seems incomplete, because one of the 15 miRNAs was found to be downregulated in patients with MDD, which does not correspond to the stated conclusion that all 15 miRNAs have increased plasma levels in the disease group.

Comments on the Quality of English Language

The manuscript is written in average quality English that needs improvement and partially conforms to an academic writing style.

Author Response

Dear reviewers, 

Thank you for pointing out our shortcomings! We have worked on revising our manuscript, and, as required, we have made the following changes: 

- Abstract: re-wrote it to better summarize the whole manuscript - intoduction, aim, methods, results and discussions. 

- Introduction: we considered trimming it to make it more precise and on point. First we introduced the concept of MDD, then we introduced RNAs and miRNAs, and then we wrote about the potential connection between miRNA dysregulation and MDD while stating the aim of the study. 

- Materials and Methods: we presented this section in a more clear and precise way - study design and population - miRNA manipulation - statistical analysis. We tried to explain the methodology better than the previous manuscript. 

- Results: we made major changes in this section. We structured it differently, in 3 subsections, each with a clear purpose: short summary of the study population - t-test results - ROC curve results. In order to prove data accuracy, tables were provided across all subsections. 

- Discussions: significant changes have been made in this section too. We discussed our results, specifically the miRNAs that showed significant dysregulation in our study, and how our results correlate with other studies. We added two paragraphs about the limitations of our study. 

- Conclusions: we believe that this section is now more concise and better summarizes our findings. 

Overall, we made significant changes to the language we used, in order to comply to academic writing standards. We proof-read the manuscript twice in order to remove any typographic errors. 

While this version of the manuscript may still need revision, we hope that this is an improvement. 

In the name of the authoring team, and as the submitting author, I would like to present our gratitude for helping us turn this manuscript into a good piece of medical literature! Thank you! 

Kind regards, 

Norberth Varga

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

congratulations on your manuscript. I would like to propose several modifications to enhance the clarity and scientific rigor of your work

1.      Introduction:

- Line 51 and line 55: could you please change the phrase “well known” as it is repeated?

  - The section is lengthy and contains a lot of repetition, especially in the description of miRNA biogenesis and classification, which diverts focus from the study's aim (Lines 66–84).

  - The aim of the study is not sufficiently clear until Line 37. A more explicit statement about the novelty and significance of the research should be introduced earlier.

  - While genetic and environmental influences are discussed (Lines 60-64), further elaboration on how these factors integrate with miRNA changes specific to MDD is lacking. A clearer transition is needed to guide the reader into the main study topic.

  Line 154: the number of the page is missing from the right side and it appears half, in the left. Could you please fix it?

2. Materials and Methods:

  - The sample size is small (10 patients and 8 controls; Lines 158-159), which is a significant limitation and raises concerns about the generalizability of the findings. This needs to be addressed explicitly.

  - The statistical methodology does not justify whether power analysis was conducted to determine an adequate sample size (Line 150).

  - Ethical approvals and informed consent are properly mentioned (Lines 135–136), but more details about patient demographics could provide clarity on sample heterogeneity.

3. Results:

  - There is no sufficient explanation for the clinical significance of the identified miRNAs, particularly hsa-miR-532-5p, which shows contradictory behavior compared to previous studies (Lines 182-185).

  - The results should better highlight the novelty of these specific miRNAs in relation to MDD in comparison to other similar studies (Lines 169-176).

  - The description of results lacks details on why certain miRNAs, such as hsa-miR-320c, show significant variation but are not fully explored (Lines 246–249). The use of independent t-tests for such a small sample may also be underpowered, warranting caution in drawing conclusions.

4. Discussion:

  - A stronger critical analysis of the limitations, especially the small sample size, and statistical power, is needed (Lines 309–314).

  - The novelty of the study should be emphasized more clearly. Some of the references to miRNAs in MDD, such as hsa-miR-532-5p (Lines 280–285), are presented without a sufficient critical stance on how these findings differ or add to previous research.

Thank you

 

 

Author Response

Dear reviewers, 

Thank you for pointing out our shortcomings! We have worked on revising our manuscript, and, as required, we have made the following changes: 

- Abstract: wrote it again to better summarize the whole manuscript - intoduction, aim, methods, results and discussions. 

- Introduction: we considered trimming it to make it more precise and on point. First we introduced the concept of MDD, then we introduced RNAs and miRNAs, and then we wrote about the potential connection between miRNA dysregulation and MDD while stating the aim of the study. 

- Materials and Methods: we presented this section in a more clear and precise way - study design and population - miRNA manipulation - statistical analysis. We tried to explain the methodology more concisely and clearly than the previous version of the manuscript. The statistical analysis consists of normality of data assessment, t-test and ROC curve analysis. the Shapiro-Wilk or Kolmogorov-Smirnov tests could not be performed due to the small sample size. 

- Results: we made major changes in this section. We structured it differently, in 3 subsections, each with a clear purpose: short summary of the study population - t-test results - ROC curve results. In order to prove data accuracy, tables were provided across all subsections. The reason we only have 10 patients  was addressed and it is stated in the first paragraph: only 10 patients have signed an informed consent while data collection took place (September 2023). 

- Discussions: significant changes have been made in this section too. We discussed our results, specifically the miRNAs that showed significant dysregulation in our study, and how our results correlate with other studies. We added two paragraphs about the limitations of our study and we clearly stated that further research is needed to validate our results and to ensure their applicability to a broader population. 

- Conclusions: we believe that this section is now more concise and better summarizes our findings. 

Overall, we made significant changes to the language we used, in order to comply to academic writing standards. We proof-read the manuscript twice in order to remove any typographic errors. 

While this version of the manuscript may still need revision, we hope that this is an improvement. Please let us know if we can improve it further.

In the name of the authoring team, and as the submitting author, I would like to present our gratitude for helping us turn this manuscript into a good piece of medical literature! Thank you! 

Kind regards, 

Norberth Varga

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This version of the manuscript has been revised in accordance with the reviewer's comments. The article can be recommended for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your thorough work in responding to my comments. I appreciate the clarity and detail with which you addressed my concerns and made the requested revisions. Your effort to improve the manuscript by incorporating the suggested clarifications and additions is commendable.

I have no additional commnents.

Best regards