MiR-199a-3p Regulates the PTPRF/β-Catenin Axis in Hair Follicle Development: Insights into the Pathogenic Mechanism of Alopecia Areata

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors write a very interesting article about candidate molecules through RNA-se-80 sequencing that may be associated with hair follicle development in lambs and explore 81 the underlying mechanisms using in vitro and in vivo models. In my opinion in the Introduction, the authors should write more information about factors leading to alopecia. Especially about molecular mechanisms leading to this disease.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Editor:

 

In accordance with your request, we have carefully revised the manuscript, and the following is our response to your request point by point.

 

Editor comments
Q1: Comments on the Quality of English Language; Minor editing of English language required.

A1: According to your evaluation, we have further optimized the language of the manuscript. Additionally, necessary supplements have been made to the introduction section.

Reviewer 2 Report

Comments and Suggestions for Authors

In their manuscript, Wang and collegues aim at identifying the effects of miR-199a-3p in hair follicle development in a lamb model.

 

The introduction does not propose any detail in regards to miR-199a-3p. Why was this miRNA used? Is there any previous data on it and its role in hair follicle development/skin? Please make a paragraph about this, to justify the question in the manuscript.  If you only choose this because your high-throughput miRNA-sequencing was referring to this gene, then indicate in the introduction that you performed a hight throughput screen and based ont hat you choose miR-199a-3p. Otherwise I would rephrase the last paragraph, so that the aim was to screen what miRNAs are involved in the process and establish a model using one of them miR-199a-3p.

 

In the Results, the authors describe that fine wool tissues were upregulating miR199-3p compared to coarse wool tissues. What is the difference between the two lamb types? Is there a genetic differenece? Is there a higher susceptibility to any hair-follicle development problem of any of these lambs? I would need some explanation and justification in here or in the introduction to enlight the methodology and the why behind the methodology.

 

Results 2.2 How stable are the miRNA mimics? What amount is still there after 6 days of culturing, how did you choose this time point?

 

Results 2.3 I do not understand the rationale to inject mir-199a-3p mimics into the fine wool lambs, if they already express i tat a higher level than the coarse-wool group. Could you explain this in the paper?

 

Results 2.4 The text says that miR-199a-3p agomirs were used, however the title and the figure 5 A indicates the use of mir-199a-3p antagomirs. While in Figure 5A antagomirs are written, but agomirs are written in the text. I am sure this is just a mistake, but please correct it, because it is very confusing.

 

In the discussion, the authors claim, that the main interest is the activation of PTír142 ß-catenin through miR199a-3p and PTPRF, however, the results only mildly agree with this. I miss however discussion ont he role of miR-199a-3p, which is really the focus of the results. I think the discussion could put the results more in context and should be less vague about the role of these pathways and include more details.

 

Overall the study is interesting, however, many point are missing to be easily accessible for readers. I suggest major revision based ont he above mentioned points.

Comments on the Quality of English Language

Minor errors, but at some point text and figure are mismatched.

Author Response

Dear Editor:

 

In accordance with your request, we have carefully revised the manuscript, and the following is our response to your request point by point.

 

Editor comments
Q1:The introduction does not propose any detail in regards to miR-199a-3p. Why was this miRNA used? Is there any previous data on it and its role in hair follicle development/skin? Please make a paragraph about this, to justify the question in the manuscript.  If you only choose this because your high-throughput miRNA-sequencing was referring to this gene, then indicate in the introduction that you performed a hight throughput screen and based ont hat you choose miR-199a-3p. Otherwise I would rephrase the last paragraph, so that the aim was to screen what miRNAs are involved in the process and establish a model using one of them miR-199a-3p.

A1: The reviewer's suggestion is excellent. In accordance with the reviewer's advice, we have provided additional clarification in the last paragraph of the introduction section.

 

Q2:In the Results, the authors describe that fine wool tissues were upregulating miR199-3p compared to coarse wool tissues. What is the difference between the two lamb types? Is there a genetic differenece? Is there a higher susceptibility to any hair-follicle development problem of any of these lambs? I would need some explanation and justification in here or in the introduction to enlight the methodology and the why behind the methodology.

A2: As the name implies, coarse and fine wool lambs primarily differ in the diameter of the wool fibers. These two can serve as a contrast to each other. The elevated expression of miR199-3p in the fine wool group indicates a correlation in two aspects. Firstly, the expression of miR199-3p is associated with follicle development. Secondly, the expression of miR199a-3p may be related to the fine wool type. This is also the reason why we chose to conduct mouse and lamb model experiments later on.

     These two types of lambs share a half-sibling genetic background.

     In fact, we conducted a series of studies on these two types of lamb models from different perspectives in the early stages, with the functional role of miR-199a-3p being one of the research directions. In our previously published articles, both types of lambs belong to fine wool breeds at the lamb stage, with only differences in fleece type, and they both develop into fine wool with consistent fiber thickness in adulthood [1,2,3]. Moreover, in our earlier publications, we focused on investigating the factors contributing to the coarse wool trait, including a cluster of miRNAs in the Gtl2 region and key DNA methylation-related sites [1,2]. In this study, we explore the impact of miR-199a-3p on the fine wool trait and its significance in the research of alopecia areata from the perspective of a fine wool model.

 

 

 

 

Q3:Results 2.2 How stable are the miRNA mimics? What amount is still there after 6 days of culturing, how did you choose this time point?

A3: Our experiments indicate that miR-199a-3p mimics reach peak expression 48 hours post-transfection, gradually diminishing thereafter. By day 6, although the expression of miR-199a-3p remains significantly different compared to the control group, the fold change in differences is not as prominent as during the peak expression period. The series of results presented in this manuscript are based on extensive preliminary experiments. We conducted pre-experiments at 48 hours, 72 hours, 144 hours (6 days), and 168 hours post-transfection. Although the expression of the target miRNA is highest at 48 hours post-transfection, the downstream signaling pathway miR-199a-3p/PTPRF/beta-catenin is not significantly activated. The optimal time point is 144 hours (6 days) post-transfection, as evidenced by mRNA sequencing of cells at this time point, which shows a significant activation of the beta-catenin signaling pathway. This also reflects a certain lag in the activation of signaling pathways and their corresponding functions in response to the target miRNA. I believe that if this response is seen by more researchers, it could be helpful for them in conducting similar transfection experiments and functional studies, avoiding excessive preliminary experiments.

 

Q4: Results 2.3 I do not understand the rationale to inject mir-199a-3p mimics into the fine wool lambs, if they already express i tat a higher level than the coarse-wool group. Could you explain this in the paper?

A4: I am pleased to elucidate to the reviewers the rationale behind conducting intravenous injection experiments in lambs. Through omics screening, miR-199a-3p emerged as the only small RNA significantly overexpressed in the fine wool lamb group. We hypothesize that this small RNA may play a promoting role in the formation of fine wool traits. Therefore, we speculated whether overexpression of miR-199a-3p in fine wool lambs would further enhance the phenotypic expression of fine wool traits. Consequently, we selected a more potent agomir for in vivo experiments in lambs. Our findings indicate that, compared to mimics, miR-199a-3p agomir persists long-term in lamb skin tissue, and the overexpression of miR-199a-3p indeed has a further promoting effect on fine wool. Simultaneously, this indirectly reflects the promoting effect of miR-199a-3p on follicle development. On the other hand, the establishment of this model serves as a validation of the previous omics screening results.

 

Q5: Results 2.4 The text says that miR-199a-3p agomirs were used, however the title and the figure 5 A indicates the use of mir-199a-3p antagomirs. While in Figure 5A antagomirs are written, but agomirs are written in the text. I am sure this is just a mistake, but please correct it, because it is very confusing.

A5: Symptoms resembling alopecia areata only appeared at the injection site of miR-199a-3p antagomir, which is why the emphasis on antagomirs is present in the title of Results 2.4. Agomirs have a promoting effect on follicle development, whereas antagomirs exert an inhibitory effect on follicle development. The two can serve as reciprocal controls. Figure 5 includes both the agomir experimental group and the antagomir group, along with their respective control groups, providing a comprehensive comparison.

 

Q6: In the discussion, the authors claim, that the main interest is the activation of PTír142 ß-catenin through miR199a-3p and PTPRF, however, the results only mildly agree with this. I miss however discussion ont he role of miR-199a-3p, which is really the focus of the results. I think the discussion could put the results more in context and should be less vague about the role of these pathways and include more details.

A6: In accordance with the reviewer's suggestion, we have made corresponding supplements in the discussion section.

 

Q7: Overall the study is interesting, however, many point are missing to be easily accessible for readers. I suggest major revision based ont he above mentioned points.Comments on the Quality of English Language

Minor errors, but at some point text and figure are mismatched.

A7: We have addressed and revised each point in response to the reviewer's comments. Additionally, we have further refined the language in the manuscript as per the reviewer's suggestions.

 

Reference

1. Wang, J.; Hua, G.; Chen, J.; Cui, K.; Yang, Z.; Han, D.; Yang, X.; Dong, X.; Ma,

Y.; Cai, G. et al. Epigenetic mechanism of Gtl2-miRNAs causes the primitive sheep characteristics  found in purebred Merino sheep. Cell Biosci. 2023, 13, 190, doi:10.1186/s13578-023-01142-z.

2. 2. Wang, J.; Hua, G.; Cai, G.; Ma, Y.; Yang, X.; Zhang, L.; Li, R.; Liu, J.; Ma, Q.;

Wu, K. et al. Genome-wide DNA methylation and transcriptome analyses reveal the key gene for  wool type variation in sheep. J. Anim. Sci. Biotechnol. 2023, 14, 88, doi:10.1186/s40104-023-00893-6.

3. 3. Wang, J.; Hua, G.; Yang, X.; Zhang, L.; Ma, Y.; Ma, Q.; Li, R.; Wu, K.; Zhao, Y.; Deng, X. A newly identified small tRNA fragment reveals the regulation of different wool  types and oxidative stress in lambs. Sci. Rep. 2023, 13, 10213, doi:10.1038/s41598-023-36895-7.

Reviewer 3 Report

Comments and Suggestions for Authors

Ms-ijms-2695007 can be accepted with minor revisions.

Although the manuscript is well presented in its parts, some additions are necessary.

The introductory part is presented in an exhaustive manner and supported by a relevant and relatively recent bibliography. The experimental design is well articulated with both in vitro and in vivo experimental tests, which follow a clearly described methodology. The discussion and conclusions part could be integrated.

- A change is requested in the quality of the images presented, in particular of figures 5 and 6 where some diagrams have overlapping and difficult to read writing.

- Information on human genome websites could be collected in a sorted table.

- Information on primers used in real-time PCR is missing. The sequences should be presented complete with pb, TA, number of cycles, and GenBank access number, where present.

- The authors indicate the use of animals of the same sex but do not give indications of whether male or female, both in mice and in lambs. It is possible there is a difference in the response to treatment in different males or females.

-Regarding the treatment of animals with miR-199a-3p, on what basis was the dosage chosen and used?

The text must be rechecked for the presence of some errors in form and typing.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Editor:

 

In accordance with your request, we have carefully revised the manuscript, and the following is our response to your request point by point.

 

Editor comments
Q1:The introductory part is presented in an exhaustive manner and supported by a relevant and relatively recent bibliography. The experimental design is well articulated with both in vitro and in vivo experimental tests, which follow a clearly described methodology. The discussion and conclusions part could be integrated.

A1: Thank you to the reviewer. We have provided additional details in the discussion section. Due to the formatting requirements of the journal, it is necessary to describe the discussion and conclusion sections separately.

 

Q2: A change is requested in the quality of the images presented, in particular of figures 5 and 6 where some diagrams have overlapping and difficult to read writing.

A2: We have higher-resolution images available, and we will upload them during the subsequent editing and review process.

 

Q3: Information on human genome websites could be collected in a sorted table.

A3: Your suggestion is highly appreciated. In accordance with your request, we have organized the information in Table S2.

 

Q4: Information on primers used in real-time PCR is missing. The sequences should be presented complete with pb, TA, number of cycles, and GenBank access number, where present.

A4: We have provided primer-related information in Table S1.

 

Q5: The authors indicate the use of animals of the same sex but do not give indications of whether male or female, both in mice and in lambs. It is possible there is a difference in the response to treatment in different males or females.

A5: The mice and lambs used in our study were all females, and I have provided explanations at the corresponding positions in the original manuscript.

 

Q6: Regarding the treatment of animals with miR-199a-3p, on what basis was the dosage chosen and used?

A6: Regarding the dosage of miR-199a-3p, our miR-199a-3p agomir and antagomirs were purchased from RiboBio Co., Ltd. in China. The dosage for mice was determined based on the maximum recommended local administration dosage provided in the relevant product instructions (recommended dose: 1-10 nmol). For lambs, due to their larger body weight, we conducted experiments using a dosage that was 10 times the maximum recommended dosage for mice, as suggested by the reagent company. The observed effects met the expectations of the experiments.

 

Q7: The text must be rechecked for the presence of some errors in form and typing. Comments on the Quality of English Language. Minor editing of English language required

A7: We carefully reviewed the content and format of our manuscript. We have further refined the language in the manuscript as per the reviewer's suggestions.