In Vitro and in Silico Analysis of miR-125a with rs12976445 Polymorphism in Breast Cancer Patients
, Joanna Miskiewicz 2, Natalia Szostak 3, Marta Szachniuk 2,3,*, Sylwia Grodecka-Gazdecka 4 and Paweł P. Jagodziński 1
Round 1
Reviewer 1 Report
Authors investigated two variants of rs12976445 polymorphism of miR-125a in blood samples from breast cancer and control patients. The good number of samples was used (175 blood samples from breast cancer patients and 129 control samples), although no significant association was found. Using single nucleotide polymorphism (SNP) analysis, higher frequency of the TT genotype was found among BC samples / HER2 positive patients. Authors also used In silico modelling to assess the presence of uridine (U) and found diminished probability of pri-miR-125a binding to NOVA1 and 26 HNRNPK proteins associated with presence of U. Authors concluded that U an C -variants could promote different RNA folding patterns and provoke alternative protein binding associated with a lower miR-125a expression in breast cancer.
Despite statistical tests indicated only a tendency, I think that this data can be used in future meta-analysis and study deserves to be published.
The study is interesting and delivers reliable data.
However, there are several issues to address.
- The fact that you observed only a tendency ( and no significant association) should be clearly reflected in the Abstract.
- Discussion section requires re-structuring: it is necessary to start with description of your own findings and support them with the previous data. Therefore, you need to move down two first paragraphs and move up the 3rd paragraph which starts with line 294.
- To increase the value of this study, authors should design and provide a schematic presentation of miR-125a signaling targets/ and hypothetical binding proteins.
- Lin 352: you need to indicate that larger breast cancer study is required to confirm the finding and test the significance of the observed associations.
Minor mistakes:
Line 60: re-phrase this sentence (replace “what is more” with “Furthermore” or just delete)
Line 114 – clean up mistypes (2.6..Predicting…)
Line 266 – replace “ suggest SNP-dependence folding ..” with “suggest SNP-dependent folding”.
Author Response
Reviewer 1: The fact that you observed only a tendency (and no significant association) should be clearly reflected in the Abstract.
Answer: We agree with this comment. The Abstract has been revised as suggested.
Former version
P.1 L.29-30: We found a higher frequency of the TT genotype in the first group, especially among HER2 positive patients, compared to control samples.
Corrected version
P.1 L.29-32: We observed the tendency that in breast cancer cases TT genotype appeared slightly more frequent over CC and CT genotypes (statistically nonsignificant). The TT genotype appeared also to be more frequent among HER2 positive patients, compared to HER2 negative.
Reviewer 1:Discussion section requires re-structuring: it is necessary to start with description of your own findings and support them with the previous data. Therefore, you need to move down two first paragraphs and move up the 3rd paragraph which starts with line 294.
Answer: According to suggestions, we have restructured the Discussion section. The paragraph which started with line 295 has been moved to the beginning of the Discussion section. We have also corrected the first sentence of a new first paragraph in order to make it clearer. Moreover, we have added more explanation after the first new sentence.
Former version
P.10 L.295-297: The analysis of our results revealed that the TT genotype was slightly more frequent in breast cancer patients and HER2 positive patients.
Corrected version
P.10 L.288-289: The analysis of our results concerning rs12976445 SNP in miR-125a revealed that the TT genotype was slightly more frequent in breast cancer patients and HER2 positive patients.
Reviewer 1:To increase the value of this study, authors should design and provide a schematic presentation of miR-125a signaling targets/ and hypothetical binding proteins.
Answer: Following the suggestion, we have drawn a supplementary figure with the caption. The panel (a) with 125a signalling targets and panels (b) and (c) with hypothetical binding proteins.
Reviewer 1: Lin 352: you need to indicate that larger breast cancer study is required to confirm the finding and test the significance of the observed associations.
Answer: In accordance with the suggestion of the Reviewer 1, we have placed new sentences at the end of the Discussion P. 11 L. 357: “The limitation of the current study is the number of cases and controls. In the future study, a larger group of breast cancer patients should be analysed to confirm the tendency of TT genotype association with breast cancer and to test the significance of the observed associations.”
Reviewer: Line 60: re-phrase this sentence (replace “what is more” with “Furthermore” or just delete)
Answer: P.2 L.69 ‘What is more’ was removed.
Reviewer: Line 114 – clean up mistypes (2.6..Predicting…)
Answer: P.3 L.124 double dots were removed.
Reviewer: Line 266 – replace “ suggest SNP-dependence folding ..” with “suggest SNP-dependent folding”.
Answer: P.10 L.286 ‘dependence’ was replaced by ‘dependent’.
Reviewer 2 Report
Lehmann et al have investigated the impact of a miR-125a SNP in the context of breast cancer using in silico modelling, finding the SNP influences RNA folding in a 51 nt fragment formed by restriction digest of the pri-miR-125a molecule and therefore may influence mature miRNA levels.
Unsure why ref no 23 was cited - this paper describes finding miRNA binding sites in mRNA, a completely different question to this manscript, using miRanda and mfold - neither software was used here.
Representative data for the restriction digest used for genotyping should be included as suplementary data. This should include the positive controls included in this digest to ensure exclusivity of the restriction enzyme working only on the C variant and not the T as suggested in the manuscript.
Nomenclature is not consistent between text and within table 1 (i.e. CT+CC and C/T-C/C)
In table 3 and 4 nothing is bold to indicate SNP location as suggested in table title.
When discussing the RNA fold figures text includes descriptions such as A1.1, A1.2, B2, B2 - what does this refer to? The labels should be consistent with the figure presented.
Are there SNPs within the other miRNAs in the miR-125a cluster (miR-99b and Let-7e) that correlate to the miR-125a variant?
Author Response
Dear Reviewer,
We would like to resubmit the Manuscript ID: applsci-959227 after an edition made strictly according to the suggestions of the Reviewers.
Thank you very much for consideration of our work for a publication in Special Issue of Applied Sciences. We greatly appreciate all the comments from the Reviewers. Considering these comments as very constructive, we edited the manuscript following the Reviewers’ suggestions.
Please find below our detailed responses to the Reviewers’ comments. We also attach a new version of the manuscript with modifications visible in the word tracking system. According to the suggestion of the Reviewers, we prepared two supplementary figures: Figure 1S with restriction digestion explained in the separate capture file, and Figure 2S with a schematic presentation of miR-125a signaling targets and hypothetical binding proteins revealed using computational analysis.
Please see the attachment.
Author Response File: 
Author Response.doc
Reviewer 3 Report
The manuscript entitled "In vitro and in silico analysis of miR-125a with rs12976445 polymorphism in breast cancer patients" describes a very interesting research area within miR-125a and breast cancer.
I suggest accepting the paper after the following minor revisions:
- All the presenting p-values have no significant results. Highlight and discuss why it´s not significans.
- It´s not clear how the control differe from the patien samples.
- It´s not clear if the control samples have any other cancer types or diseases.
- Go thrue the text, some misspelling here and there.
Author Response
Reviewer 3
Reviewer 3: All the presenting p-values have no significant results. Highlight and discuss why it´s not significans.
Answer: We agree with this comment and we address this problem in the new version of the manuscript. We have inserted the following sentences to Discussion P.10 L.290-295: “Our study revealed only a tendency, and we obtained P values above 0.05. One reason is the participation of miR-125a with numerous other factors in the development of breast cancer. The correlation of such multigenic diseases with single SNP requires a greater number of individuals and controls. Our findings suggested that rs12976445 has the potential to be a predictive biomarker for cancer risk, but a meta-analysis of a greater number of cases is required.”
Reviewer: It´s not clear how the control differe from the patien samples.
Answer: According to the suggestion of the Reviewer 3 the description of controls has been edited. The only difference between controls and cases was a negative diagnosis concerning breast cancer. The women were in the same age range.
Former version:
P.2 L.86-88. 175 blood samples were obtained by antecubital vein puncture. 129 control blood samples were collected from patients diagnosed negatively for breast cancer. All 304 samples were collected from patients of the Department of Surgery, Chair of Oncology at PUMS.
Corrected version:
P.2 L.88-91. 175 blood samples were obtained by antecubital vein puncture. 129 control blood samples were collected from patients diagnosed negatively for breast cancer, and not diagnosed with other types of cancer. All 304 samples were collected from 14-88 years old patients of the Department of Surgery, Chair of Oncology at PUMS.
Reviewer 3: It´s not clear if the control samples have any other cancer types or diseases.
Answer: The samples were collected from patients who were assessed as being under risk of breast cancer. The samples were collected and classified as controls when the diagnose of breast cancer was negative. However, we cannot exclude other diseases in control patients.
Reviewer 3: Go thrue the text, some misspelling here and there.
Answer: Thank you for the suggestion. We extensively edited the manuscript and we corrected all recognised misspelling.
Round 2
Reviewer 2 Report
Authors have made changes to the manuscript which have addressed my comments/queries.
