Analysis and Identification of miRNA in Skin Tissues of Tibetan Cashmere Goats (Capra hircus) with Different Cashmere Fineness
FU Xue-Feng1, ZHAO Bing-Ru2, SUO Lang-Da3, BA Gui3, DE Ji3, AWANG Cuo-Ji3, WU Yu-Jiang3,*, TIAN Ke-Chuan4,*
1 Key Laboratory of Genetics Breeding and Reproduction of Xinjiang Wool-sheep & Cashmere-goat/Institute of Animal Science, Xinjiang Academy of Animal Sciences, Urumqi 830011, China; 2 College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; 3 Institute of Animal Science, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850009, China; 4 Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China
Abstract：As an important gene regulatory element, microRNA (miRNA) is expressed in various mammalian skin and hair follicles, regulating the growth and development of skin and hair at post-transcriptional level. The aim of present study was to analyze and identify miRNAs in the skin tissues of Tibetan cashmere goats, and explore the role and regulatory mechanisms of miRNAs in fiber diameter traits of Tibetan cashmere goats (Capra hircus). Four Tibetan cashmere goats with fineness cashmere (average fiber diameter (12.04±0.03) μm) and 4 goats with coarse cashmere (average fiber diameter (14.88±0.05) μm) were selected, respectively. Shoulder skin tissues were collected to extract RNA and established libraries for high-throughput sequencing. After the raw sequences were filtered and blasted with reference genome (ARS1), the miRNAs and other small RNAs were identified based on the known miRNAs in miRBase and the location information of other non-coding RNAs (ncRNAs) in the reference genome. The differentially expressed miRNAs in the skin tissues of Tibetan cashmere goats with different fiber diameters were screened out, and target-gene prediction and function analysis were performed. qPCR was used to detect the consistency of 5 miRNAs with the sequencing results. The results showed that each sample obtained at least 20.09 M raw reads and at least 19.46 M clean reads after filtering. A total of 426 known miRNAs and 119 novel miRNAs were identified. Twelve differentially expressed miRNAs were screened, of which 5 miRNAs were significantly up-regulated in the skin tissues of the goats with fineness cashmere when compared with that of coarse group, and 7 miRNAs were significantly down-regulated. Differentially expressed miRNAs were predicted to obtain a total of 621 target genes. KEGG enrichment analysis showed that the target genes were enriched in biological pathways such as B cell receptor signaling pathway, FcεRI signaling pathway, and T cell receptor signaling pathway. Meanwhile, qPCR results showed that the expression trends of selected 5 miRNAs were consistent with that of the sequencing results. The above results suggests the fineness of Tibetan cashmere might be regulated by different miRNAs, which further affecting the expression of target genes and simultaneously participating in the regulation of multiple signaling pathways. The present study provides a reference for deep researches on miRNAs and related signaling pathways regulating the fineness of cashmere.
付雪峰, 赵冰茹, 索朗达, 巴贵, 德吉, 阿旺措吉, 吴玉江, 田可川. 不同绒细度的西藏绒山羊皮肤组织miRNA分析与鉴定[J]. 农业生物技术学报, 2021, 29(11): 2118-2128.
FU Xue-Feng, ZHAO Bing-Ru, SUO Lang-Da, BA Gui, DE Ji, AWANG Cuo-Ji, WU Yu-Jiang, TIAN Ke-Chuan. Analysis and Identification of miRNA in Skin Tissues of Tibetan Cashmere Goats (Capra hircus) with Different Cashmere Fineness. 农业生物技术学报, 2021, 29(11): 2118-2128.
[1] 白宇. 2018. Tβ4通过作用于JNK和Notch信号通路影响毛囊生长的机制研究[D]. 硕士学位论文, 内蒙古大学, 导师: 郭旭东, pp. 44-45. (Bai Y. 2018. The mechanism of Tβ4 regulating on the hair growth through JNK and NOTCH signaling pathways[D]. Thesis for M.S., Inner Mongolia University, Supervisor: Guo X D, pp. 44-45.) [2] 次仁德吉. 2012. 藏西北地区绒山羊产绒性能研究[D]. 硕士学位论文, 中国农业科学院, 导师: 马月辉, pp. 34-35. (Cirendeji. 2012. The study of cashmere performance in northwest tibet[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Ma Y H, pp. 34-35.) [3] 樊凯军. 2014. 绒山羊皮肤microRNA的研究[D]. 硕士学位论文, 内蒙古农业大学, 导师: 尹俊, pp. 35-36. (Fan K J. 2014. Study on skin microRNAs in cashmere goat[D]. Thesis for M.S., Inner Mongolia Agricultural University, Supervisor: Yin J, pp. 35-36.) [4] 马涛. 2019. miRNA-203在绒山羊毛囊发育周期中的差异表达及其靶基因功能鉴定[D]. 硕士学位论文, 吉林大学, 导师: 张巧灵, pp. 47-48. (Ma T. 2019. Differential expression of miRNA-203 in hair follicle development cycle of cashmere goats and identification of target gene function[D]. Thesis for M.S., Jilin University, Supervisor: Zhang Q L, pp. 47-48.) [5] 张桂山. 2016. 辽宁绒山羊与乾华肉用美利奴羊皮肤毛囊miRNA筛选及靶基因验证[D]. 博士学位论文, 吉林农业大学, 导师: 姜怀志, pp. 78-79. (Zhang G S. 2016. Screening of miRNAs and identification of candidate target genes in skin and hair follicle from liaoning cashmere goat and qianhua mutton merino[D]. Thesis for Ph.D., Jilin Agricultural University, Supervisor: Jiang H Z, pp. 78-79.) [6] 郝玉, 尹俊. 2014. 绒山羊70 d胚胎与新生羊羔体侧部皮肤miRNA的比较分析[J]. 生物技术, 24(5): 71-76. (Hao Y, Yin J. 2014. Analysis of miRNAs between 70 days fetal and lamb skin[J]. Biotechnology, 24(5): 71-76.) [7] 唐晓惠, 柳广斌, 杜小勇, 等. 2012. 藏绵羊毛囊组织中microRNA-31的表达量变化[J]. 农业生物技术学报, 20(10): 1178-1183. (Tang X H, Liu G B, Du X Y, et al. 2012. Expression variation of microRNA-31 in wool follicle of tibetan sheep, 20(10): 1178-1183.) [8] 李阳, 刘文天. 2011. 微小RNA与DNA甲基化相互调控在胃癌中的作用[J]. 国际肿瘤学杂志, 38(9): 684-687. (Li Y, Liu W T. 2011. Mutual regulation between miRNA and DNA methylation in gastric cancer[J]. Journal of International Oncology, 38(9): 684-687.) [9] 李转见, 韩瑞丽, 蓝贤勇, 等. 2013. 山羊miRNA研究进展[J]. 家畜生态学报, 34(8): 6-10. (Li Z J, Han R L, Lan X Y, et al. 2013. Research progress of caprine miRNAs[J]. Journal of Domestic Animal Ecology, 34(8): 6-10.) [10] 吴月红, 李勇, 杨易, 等. 2016. microRNAs在动物皮肤及毛囊发育中的调控作用研究进展[J]. 中国畜牧兽医, 43(9): 2373-2378. (Wu Y H, Li Y, Yang Y, et al. 2016. Research progress on regulation of microRNAs in animal skin and hair follicle[J]. China Animal Husbandry and Veterinary Medicine, 43(9): 2373-2378.) [11] 吴玉江, 付雪峰, 索朗达, 等. 2020. 西藏绒山羊WNT4和HOXC13基因对绒毛纤维直径性状的遗传效应分析[J]. 中国畜牧兽医, 47(2): 497-505. (Wu Y J, Fu X F, Suo L D, et al. 2020. Genetic effect analysis of WNT4 and HOXC13 gene on fiber diameter trait in Tibetan cashmere goats[J]. China Animal Husbandry and Veterinary Medicine, 47(2): 497-505.) [12] 吴玉江, 益西多吉, 索朗达, 等. 2013. 西藏绒山羊产业现状及发展对策[J]. 家畜生态学报, 34(9): 82-85. (Wu Y J, Yixiduoji, Suo L D, et al. 2013. Current situation and development strategies of Tibetan cashmere industry[J]. Journal of Domestic Animal Ecology, 34(9): 82-85.) [13] 吴玉江, 益西多吉, 索朗达, 等. 2014. 西藏绒山羊本品种选育效果研究[J]. 畜牧与兽医, 46(9): 38-41. (Wu Y J, Yixiduoji, Suo L D, et al. 2014. Study on the breeding effect of tibetan cashmere goats[J]. Animal Husbandry and Veterinary Medicine, 46(9): 38-41.) [14] 俞焙秦, 刘炳亚. 2007. miRNA的生物学特性和功能[J]. 上海交通大学学报(医学版), 27(5): 621-623. (Yu B Q, Liu B Y. 2007. Biological character and function of miRNA[J]. Journal of Shanghai Jiaotong University (Medical Science), 27(5): 621-623.) [15] 赵爽, 刘默芳. 2009. MicroRNA作用机制研究的新进展[J]. 中国科学(C辑: 生命科学), 39(1): 109-113.., 39(1): 109-113.) [16] Aubin-Houzelstein G.2012. Notch signaling and the developing hair follicle[J]. Advances in Experimental Medicine and Biology, 727: 142-160. [17] Bhattacharya S, Wheeler H, Leid M, et al.2015. Transcription factor CTIP2 maintains hair follicle stem cell pool and contributes to altered expression of LHX2 and NFATC1[J]. The Journal of Investigative Dermatology, 135(11): 2593-2602. [18] Burnside J, Ouyang M, Anderson A, et al.2008. Deep sequencing of chicken microRNAs[J]. BMC Genomics, 9(1): 185-193. [19] Dai B, Hao F, Xu T, et al.2020. Thymosin β4 identified by transcriptomic analysis from HF anagen to telogen promotes proliferation of SHF-DPCs in albas cashmere goat[J]. International Journal of Molecular Sciences, 21(7): 2268-2282. [20] Forstbauer L M, Brockschmidt F F, Moskvina V, et al.2012. Genome-wide pooling approach identifies SPATA5 as a new susceptibility locus for alopecia areata[J]. European Journal of Human Genetics, 20(3): 326-332. [21] Fu X, Zhao B, Tian K, et al.2020. Integrated analysis of lncRNA and mRNA reveals novel insights into cashmere fineness in Tibetan cashmere goats[J]. PeerJ, 8: e10217. [22] Harel S, Higgins C A, Cerise J E, et al.2015. Pharmacologic inhibition of JAK-STAT signaling promotes hair growth[J]. Science Advances, 1(9): e1500973. [23] Hsu S D, Lin F M, Wu W Y, et al.2010. miRTarBase: A database curates experimentally validated microRNA-target interactions[J]. Nucleic Acids Research, 39(suppl_1): D163-D169. [24] Ito T, Tsuji G, Ohno F, et al.2016. Activation of the OVOL1-OVOL2 axis in the hair bulb and in pilomatricoma[J]. The American Journal of Pathology, 186(4): 1036-1043. [25] Janssen H L, Reesink H W, Lawitz E J, et al.2013. Treatment of HCV infection by targeting microRNA[J]. New England Journal of Medicine, 368(18): 1685-1694. [26] Koyama T, Kobayashi K, Hama T, et al.2016. Standardized scalp massage results in increased hair thickness by inducing stretching forces to dermal papilla cells in the subcutaneous tissue[J]. Eplasty, 16: e8. [27] Kozomara A, Griffiths-Jones S.2013. miRBase: Annotating high confidence microRNAs using deep sequencing data[J]. Nucleic Acids Research, 42(D1): D68-D73. [28] Langmead B, Trapnell C, Pop M, et al.2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[J]. Genome Biology, 10(3): R25. [29] Lin H-Y, Yang L-T.2013. Differential response of epithelial stem cell populations in hair follicles to TGF-β signaling[J]. Developmental Biology, 373(2): 394-406. [30] Liu G B, Liu R Z, Li Q Q, et al.2013. Identification of microRNAs in wool follicles during anagen, catagen, and telogen phases in Tibetan sheep[J]. PLOS ONE, 8(10): e77801. [31] Muhammad S A, Fatima N, Paracha R Z, et al.2019. A systematic simulation-based meta-analytical framework for prediction of physiological biomarkers in alopecia[J]. Journal of Biological Research, 26(1): 2-17. [32] Rile N, Liu Z H, Gao L X, et al.2018. Expression of vimentin in hair follicle growth cycle of inner mongolian cashmere goats[J]. BMC Genomics, 19(1): 38-45. [33] Rishikaysh P, Dev K, Diaz D, et al.2014. Signaling involved in hair follicle morphogenesis and development[J]. International Journal of Molecular Sciences, 15(1): 1647-1670. [34] Robinson M D, Mccarthy D J, Smyth G K.2009. edgeR: A bioconductor package for differential expression analysis of digital gene expression data[J]. Bioinformatics, 26(1): 139-140. [35] Robinson M D, Oshlack A.2010. A scaling normalization method for differential expression analysis of RNA-seq data[J]. Genome Biology, 11(3): R25. [36] Song S, Yao N, Yang M, et al.2016. Exome sequencing reveals genetic differentiation due to high-altitude adaptation in the Tibetan cashmere goat (Capra hircus)[J]. BMC Genomics, 17(1): 122. [37] Stocks M B, Moxon S, Mapleson D, et al.2012. The UEA sRNA workbench: A suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets[J]. Bioinformatics, 28(15): 2059-2061. [38] Troy T C, Turksen K.2007. The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities[J]. Molecular Biotechnology, 36(2): 166-174. [39] Wang E, Harel S, Christiano A M.2016. JAK-STAT signaling jump starts the hair cycle[J]. Journal of Investigative Dermatology, 136(11): 2131-2132. [40] Yao L, Bao A, Hong W, et al.2019. Transcriptome profiling analysis reveals key genes of different coat color in sheep skin[J]. PeerJ, 7(5): e8077. [41] Yuan C, Wang X L, Geng R Q, et al.2013. Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing[J]. BMC Genomics, 14(1): 511. [42] Zhang H, Su Y, Wang J, et al.2019. Ginsenoside Rb1 promotes the growth of mink hair follicle via PI3K/AKT/GSK-3β signaling pathway[J]. Life Sciences, 229: 210-218.
