The Identification and Characteristics of miRNAs Related to Cashmere Fiber Traits in Skin Tissue of Cashmere Goats
, Zhiyun Hao, Yuzhu Luo, Xiu Liu, Shaobin Li, Fangfang Zhao and Mingna LiRound 1
Reviewer 1 Report
The study was carried out to identify and reveal the properties of miRNAs that are thought to be effective on fiber properties in cashmir goats. The number of examples used is sufficient for the character to be defined. The method used in the study is suitable for the purpose of the study. In the conclusion part of the study, it was stated that 144 miRNAs that differed significantly in two different cashmere breeds were expressed, and it was stated that it would contribute significantly to future molecular genetic studies. However, it does not contain information on how to use the revealed 144 miRNAs in selection planning for fiber properties that may come into play in the future. For this reason, the results obtained in the conclusion part should also be evaluated in terms of animal breeding, where it can make an important contribution.
Author Response
The study was carried out to identify and reveal the properties of miRNAs that are thought to be effective on fiber properties in cashmir goats. The number of examples used is sufficient for the character to be defined. The method used in the study is suitable for the purpose of the study. In the conclusion part of the study, it was stated that 144 miRNAs that differed significantly in two different cashmere breeds were expressed, and it was stated that it would contribute significantly to future molecular genetic studies. However, it does not contain information on how to use the revealed 144 miRNAs in selection planning for fiber properties that may come into play in the future. For this reason, the results obtained in the conclusion part should also be evaluated in terms of animal breeding, where it can make an important contribution.
AU: We have improved the conclusions of the manuscript in lines 361-368.
Reviewer 2 Report
See comments and suggestions in attached pdf.
Comments for author File: 
Comments.pdf
Author Response
See comments and suggestions in attached pdf.
AU：We have revised these according to the suggestions and comments in the attached PDF proposed by the Reviewer.
Reviewer 3 Report
The manuscript by Qiao et al. reports on miRNA expression profiles of Liaoning cashmere goat and Ziwuling black goats. The different properties and qualities of their cashmere coats are referred to different signaling pathways highlighted by particular miRNAs.
The study is interesting and original. The analyses were done thoroughly.
However, it appears unclear to me why skin samples were investigated in this study. As the authors suggest, there are two types of hair follicle, primary hair follicle producing wool fiber and the secondary hair follicle producing cashmere fiber. Subsequently, I consider it necessary to investigate hair follicles to dissect the cashmere traits. Otherwise, the expression profiles of the two hair types are mixed and might thereby cloak important mechanisms.
Furthermore, I wonder why the authors specifically went for miRNAs to investigate the cashmere traits. To learn about the expression profiles related to cashmere traits, mRNA profiles might already be sufficient. What benefit does this specific type of analysis bring to learn more about cashmere traits? It there a pathway suggested from mRNA analyses that gives reason to investigate the role of miRNAs in this study? What is the hypothesis of this work? This has to be clearly stated in this manuscript.
Author Response
The manuscript by Qiao et al. reports on miRNA expression profiles of Liaoning cashmere goat and Ziwuling black goats. The different properties and qualities of their cashmere coats are referred to different signaling pathways highlighted by particular miRNAs.
The study is interesting and original. The analyses were done thoroughly.
However, it appears unclear to me why skin samples were investigated in this study. As the authors suggest, there are two types of hair follicle, primary hair follicle producing wool fiber and the secondary hair follicle producing cashmere fiber. Subsequently, I consider it necessary to investigate hair follicles to dissect the cashmere traits. Otherwise, the expression profiles of the two hair types are mixed and might thereby cloak important mechanisms.
AU: The hair follicle is derived from the skin. It is almost impossible to separate directly the primary hair follicle and the secondary hair follicle from the skin in vivo. However, if we collect skin tissue samples and then take them back to the laboratory to separate the secondary hair follicle from skin, the status of the skin and the secondary hair follicle are significantly different from samples collected in vivo. In this context, the secondary hair follicle samples separated in laboratory do not well reflect the state of skin and hair follicle in vivo. Given that the two cashmere goat breeds used in the study produce wool and cashmere fibers, and that their wool production performance are all poor and similar, the differences in the skin tissue almost reflect the difference in cashmere fiber performance between LC and ZB goats investigated in the study.
In view of the above-mentioned reasons, most studies used skin (instead of the secondary hair follicle) to profile the expression of mRNA and miRNA in cashmere goats [1-9].
References:
1. Liu Z, Yang F, Zhao M, Ma L, Li H, Xie Y, Nai R, Che T, Su R, Zhang Y, Wang R, Wang Z, Li J. The intragenic mRNA-microRNA regulatory network during telogen-anagen hair follicle transition in the cashmere goat. Sci Rep, 2018, 8(1): 14227. doi: 10.1038/s41598-018-31986-2.
2. Ma T, Li J, Jiang Q, Wu S, Jiang H, Zhang Q. Differential expression of miR-let7a in hair follicle cycle of Liaoning cashmere goats and identification of its targets. Funct Integr Genomics, 2018, 18(6): 701-707. doi: 10.1007/s10142-018-0616-x.
3. Bai WL, Dang YL, Yin RH, Jiang WQ, Wang ZY, Zhu YB, Wang SQ, Zhao YY, Deng L, Luo GB, Yang SH. Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles. Anim Biotechnol, 2016, 27(2): 104-12. doi: 10.1080/10495398.2015.1105240.
4. Ding Y, Xue X, Liu Z, Ye Y, Xiao P, Pu Y, Guan W, Mwacharo JM, Ma Y, Zhao Q. Expression Profiling and Functional Characterization of miR-26a and miR-130a in Regulating Zhongwei Goat Hair Development via the TGF-β/SMAD Pathway. Int J Mol Sci, 2020, 21(14): 5076. doi: 10.3390/ijms21145076.
5. Li J, Qu H, Jiang H, Zhao Z, Zhang Q. Transcriptome-Wide Comparative Analysis of microRNA Profiles in the Telogen Skins of Liaoning Cashmere Goats (Capra hircus) and Fine-Wool Sheep (Ovis aries) by Solexa Deep Sequencing. DNA Cell Biol, 2016, 35(11): 696-705. doi: 10.1089/dna.2015.3161.
6. Hu S, Li C, Wu D, Huo H, Bai H, Wu J. The Dynamic Change of Gene-Regulated Networks in Cashmere Goat Skin with Seasonal Variation. Biochem Genet, 2022, 60(2): 527-542. doi: 10.1007/s10528-021-10114-2.
7. Li J, Liu L, Zhang J, Cheng L, Ren L, Zhao Y. The expression of miR-129-5p and its target genes in the skin of goats. Anim Biotechnol, 2021, 32(5): 573-579. doi: 10.1080/10495398.2020.1730392.
8. Liu Z, Xiao H, Li H, Zhao Y, Lai S, Yu X, Cai T, Du C, Zhang W, Li J. Identification of conserved and novel microRNAs in cashmere goat skin by deep sequencing. PLoS One, 2012, 7(12): e50001. doi: 10.1371/journal.pone.0050001.
9. Liu Y, Wang L, Li X, Han W, Yang K, Wang H, Zhang Y, Su R, Liu Z, Wang R, Wang Z, Zhao Y, Wang Z, Li J. High-throughput sequencing of hair follicle development-related micrornas in cashmere goat at various fetal periods. Saudi J Biol Sci, 2018, 25(7): 1494-1508. doi: 10.1016/j.sjbs.2017.12.009.
Furthermore, I wonder why the authors specifically went for miRNAs to investigate the cashmere traits. To learn about the expression profiles related to cashmere traits, mRNA profiles might already be sufficient. What benefit does this specific type of analysis bring to learn more about cashmere traits? It there a pathway suggested from mRNA analyses that gives reason to investigate the role of miRNAs in this study? What is the hypothesis of this work? This has to be clearly stated in this manuscript.
AU: The miRNAs are a kind of endogenous non-coding RNAs. Most miRNAs down-regulate expression of the target mRNAs by complementally binding to their 3'UTR. Although miRNAs accounts for less 5% of mammalian genome, they can regulate expression of up to 60% functional genes. In this context, miRNAs are important post-transcriptional regulatory factors and play essential roles in many biological processes. Important roles of miRNAs in regulation of hair fiber traits have been reported. For example, miR-31 was involved in the hair follicle growth cycle and hair shaft formation in mouse skin [1]. The miR-22 can promote apoptosis of hair follicles and repress keratinocyte differentiation in mice skin [2]. The miR-214 inhibited inducible proliferation of keratinocytes, eventually resulting in formation of fewer hair follicle with decreased hair bulb size in mice [3]. The roles of miR-205 [4] and miR-125b [5] in the proliferation and differentiation of hair follicle stem cells have also been reported. Therefore, in order to fully explain the molecular mechanisms underlying cashmere fiber growth and development, the roles of miRNAs are often investigated in cashmere goats and hair follicle development.
However, there are few studies about the miRNA expression comparison in skin tissues between different goat breeds. In our previous study, the expression profile of mRNAs extracted from the same skin tissue sample as those used in the study has been investigated. In this context, in the study, the characteristics and roles of miRNAs in cashmere fiber performance were investigated.
We have also clearly stated the hypothesis in lines 13-14 in the manuscript, namely,
References:
1. Mardaryev AN, Ahmed MI, Vlahov NV, Fessing MY, Gill JH, Sharov AA, Botchkareva NV. Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle. FASEB J, 2010, (10): 3869-81. doi: 10.1096/fj.10-160663.
2. Yuan S, Li F, Meng Q, Zhao Y, Chen L, Zhang H, Xue L, Zhang X, Lengner C, Yu Z. Post-transcriptional regulation of keratinocyte progenitor cell expansion, differentiation and hair follicle regression by miR-22. PLoS Genet, 2015, 11(5): e1005253. doi: 10.1371/journal.pgen.1005253.
3. Ahmed MI, Alam M, Emelianov VU, Poterlowicz K, Patel A, Sharov AA, Mardaryev AN, Botchkareva NV. MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway. J Cell Biol, 2014, 207(4): 549-67. doi: 10.1083/jcb.201404001.
4. Wang D, Zhang Z, O'Loughlin E, Wang L, Fan X, Lai EC, Yi R. MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway. Nat Cell Biol, 2013, (10): 1153-63. doi: 10.1038/ncb2827.
5. Zhang L, Stokes N, Polak L, Fuchs E. Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment. Cell Stem Cell, 2011, 8(3): 294-308. doi: 10.1371/journal.pone.0096857
Round 2
Reviewer 2 Report
Thank you for your effort in improving the manuscript.
A can't download table S4, as the file name is very long. I understand the English changes, but some of them were unnecessary and in some cases, the first version was easier to read and scientifically accurate. Line 126 - Please, include a reference or a link to fastp v0.18.0. Line - 278 - The references to the other species may stay.Reviewer 3 Report
The manuscript by Qiao et al. has been significantly improved by the revisions. The reference of meReap is wrongly assigned and needs to be corrected.
