Hsa-Mir-320c, Hsa-Mir-200c-3p, and Hsa-Mir-449c-5p as Potential Specific miRNA Biomarkers of COPD: A Pilot Study

Round 1

Reviewer 1 Report

the authors present some interesting preliminary findings that may help distinguish between pre-COPD smokers from smokers who are not on the progression to develop COPD.  However, there are some changes/revisions that need to be incorporated before the paper can be accepted.

1. As this is a preliminary study (as evidenced by the phrase "A pilot study" in the title, the authors need to add the word potential or putative before miRNA in the title.
2. Is there no other reference on COPD as a leading cause of death than ref 1?
3. Need to define "Pack-years in the Methods section under "Patients."
4. Please define "GOLD" in the results section (line159).
5. Figure 1a is difficult to see the 3D arrangement of the results.  Perhaps a graph with a different angle might help?
6. Lines 188-189.  What are the three different p values referring?  
7. Line 223.  Please replace validate with another term such as "qualify" or "fit-for purpose."  One study does not validate biomarkers. 
8. Figure 3.  What are the Gene ratios?  How were they calculated.  p.adkust is not discussed in the figure legend.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

  This is an important study to understand molecular mechanism of COPD using the analysis of miRNAs in BAL samples. The authors showed up-regulation of the three miRNAs and the significant correlation with COPD severity. By using in silico analysis, they suggested the involvement of EGFR and Hippo pathway in COPD progression. The approaches are cutting-edge and logical enough to propose the next generation therapy.

 

Comments:

1. Various pathophysiological condition in patients having the exclusion criteria does some effects on the miRNA results? asthma, atopy, allergic rhinitis, auto- immune diseases, renal disorders, other malignancies, lung cancer, infectious diseases and/or using immunomodulatory drugs.
2. There are no comments on responsible cell type(s) to progress the COPD stage. This is essential knowledge to understand origin of miRNAs in BAL and its role for progression of the disease stage. Please add this part of discussion.
3. Lines 294-297: It is important to suggest that the combination of three miRNAs. We need the ROC analysis using three miRNAs in comparison with ROC using the single miRNA. This analysis would give more suggestion on the authors’ discussion.
4. Around Line 251: It is better to give comment on the down-regulated miRNAs at least several relating to pathogenesis of COPD, since miRNA is suppressing gene expression and down-regulation results in up-regulation of the target gene.
5. If you find or have data to analyze EV-miRNAs in BAL from COPD, please give comments. In another word, data of the EV fraction of BAL and the whole BAL miRNAs. Readers may be interested in the origin of miR-200c-3p, miR-449c-5p, miR-320c, and this point is crucial to discuss the possible therapeutical approaches.
6. Lines 380 to 383: There is very important discussion here, because the authors recommend less invasive sample, liquid biopsy or sputum. Observations on the difference or similarity of miRNA profiles between BAL and samples from liquid biopsy is required around this discussion. Please explain why BAL is a suitable sample for analyzing miRNA than other samples: serum, plasma etc.

 

Minor points:

 

7. Criteria of each staging of GOLD should be given in somewhere.
8. Line 86: What does mean “10 pack-years”?
9. Line 95: what does mean “healthy lung”.
10. LINE 105: 108 copies of UniSp6 RNA must be 10^8 copies if it is the samw as reference 10.
11. Lines 189 to 191: Sentence is not completed, some words lacking?
12. Line 298: Please give explanation of “CS”.
13. Figure 1a: Font sizes of remarks of x, y, z axis are too small to see. Please enlarge the font size.

Author Response

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Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dear Editor-in-Chief

Thanks for your kind invitation. With no actual or potential conflict of interest I have read the manuscript entitled “hsa-miR-320c, hsa-miR-200c-3p and hsa-miR-449c-5p as miRNA 2 biomarkers specific of COPD patients. A pilot study” by Cerón-Pisa et al. Although I found it interesting, there are some serious concerns which I have mentioned them.

Comments

Abstract and Introduction

1. Abstract is very long and must be more concise and comprehensive.

Material and method (MM)

2. Please explain inclusion and exclusion criteria.
3. The manuscript is not contain any comprehensive clinical/demographic data about patients. For example Ex-smoker must be separated from other groups.
4. Real-time RT-PCR is not clearly described.
5. DLco must be presented for all subjects.
6. The stage of disease (COPD) is critical clinical data and must be presented in “Material and Methods” section. Also, stage of COPD should be considered in statistical analysis.
7. The association between the expression level of hsa-miR-320c, hsa-miR-200c-3p, hsa-miR-449c-5p and other data such as stage of COPD, respiratory parameters etc. must be done.
8. The authors do not provide any relevant clinical information; in fact, there is no information about any respiratory infection (acute or chronic) or cancer history for all groups.
9. What normalization approach was used after RT-PCR analysis? Please provide the information and add to the methods.

Results

10. Comprehensive clinical/demographic data about patients must be presented in a table.

Discussion and Conclusion

11. More details needed to be supplied about studies than your results in the discussion part.
12. Discuss about previous similar studies (by more detail), although not in the case of other respiratory disease.
13. Authors should be more careful with the claim that they can distinguish COPD from smoker and non-smoker subjects.

Others

14. The manuscript must be extensively edited by a native English speaker.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

This is an interesting study. There are sample size and methodology issues that requires clarification from the authors. 

1) Subject selection: is based on a convenience sample of patients who undergo bronchoscopy for clinical indications i.e. haemoptysis (usually related to infections, even if cultures are negative), lung mass etc. This convenience sampling might indirectly contradict the inclusion (stable with no infection within 3 months) and exclusion criteria (i.e. lung cancer). The table on the subject characteristics is lacking in sufficient detail to assess if the case selection is appropriate, which might in turn affect the miRNA results.

2) A larger sample size will provide more reassurance on the validity of the results. This is particular so for heterogenous airway diseases. The subjects used for the "validation phase" should  instead be incorporated into the "discovery phase". Typically miRNA discovery stage will require 30 subjects in each arm, n= 5 used in the screening stage is too small. 

3) Selection / discovery panel/ heatmap

• microarray method was used is typically less sensitive whan qPCR, and hence will be particularly affected by the very small sample size.
• It is not clearly described how robust the screening method is. The authors should clarify how much total RNA they used for the miRNA microarray screen.
• BAL can have varying dilutions, depending on the procedure methods used by different doctors, and is typically more dilute compared to sputum samples. Hence it is important to state more much RNA is obtained from only 200ul of BAL. The standardization of the BAL technique should be explained in detail. 
• The other miRNAs in their heatmap that looked similar to the 3 they picked, but they completely did not discuss them. Could this be because the authors did not manage to validate the array findings in the qPCR? 
• heat map indicated hsa-miR-230c but the authors talked about hsa-miR-320c throughout the text of this manuscript. Wrong miRNA candidate will generate completely wrong gene ontology data and thus invalidate part of their findings. 

Author Response

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Reviewer 3 Report

This manuscript, entitled hsa-miR-320c, hsa-miR-200c-3p and hsa-miR-449c-5p as miRNA biomarkers specific of COPD patients. A pilot study, investigated miRNAs roles in COPD.

Major concerns:

1.The authors didnot give the data the specific to copd, three miRNAs or just one of these  did not compared  that of  others dieases

2. COPD in long will transfer to lung cancer ,  the authors should investigate these miRNAs in lung cancer cell lines and patients

3. the authors should invesitgate some big data compared their own data

4.  how about one miRNA or two miRNAs as biomarkers in copd, the authors shoud give cohort or others data

5. why these miRNAs can be as biomarkers, the mechamism should be investigated, such as targets or signal transdution

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors are unable to address my concerns sufficiently to make this manuscript suitable for publication.  

Reviewer 3 Report

The revised manuscript sounded better than before， but  the project design was still not good enough.

For example. in detction samples RNA, the authors used miR-16-5p as control, but many studies showed this mirna was in differnent expression, in stages or tissuse.