Inner Mongolian Cashmere Goat Secondary Follicle Development Regulation Research Based on mRNA-miRNA Co-analysis
Inner Mongolian Cashmere Goat Secondary Follicle Development Regulation Research Based on mRNA-miRNA Co-analysis
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基于mRNA-miRNA联合分析的内蒙古绒山羊次生卵泡发育调控研究
DOI：
10.1038/s41598-020-60351-5
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发表时间：
2020-03-11
影响因子：
4.6
通讯作者：
Li, Jinquan
中科院分区：
文献类型：
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作者：
Han, Wenjing;Yang, Feng;Li, Jinquan
内蒙古绒山羊作为畜牧业生产的重要组成部分，在动物纤维产业中占有重要地位。近年来，科学研究对毛囊周期生长的分子调控机制进行了大量探索，但对胎儿期山羊绒毛发育的研究报道较少。本研究在完成21份内蒙古绒山羊胎儿期（45 d、55 d、65 d、75 d、95 d、115 d和135 d）皮肤样本的mRNA和miRNA测序的基础上，分析了绒山羊胎儿期皮肤组织中mRNA和miRNA的表达情况。通过mRNA和miRNA数据的联合分析，筛选出与绒山羊次级毛囊发育相关的miRNA靶基因。构建过表达载体，通过双荧光素酶报告基因系统鉴定miRNA与目的基因的相互作用。采用RT-qPCR和western blot方法，在内蒙古绒山羊成纤维细胞水平验证了chi-miR-199 a-5 p和TGF-β 2的功能及其相互关系。为进一步研究miRNA及其靶基因调控皮肤毛囊发生和发育提供理论基础。结果显示，7个基因（BAMBI、SMAD 1、LTBP 1、PPP 2 R1 B、ID 4、BMP 8B和PITX 2）和7种miRNA（chi-miR-17- 5 p、chi-miR-125 b-3 p、chi-miR-21- 5 p、chi-miR-143- 5 p和chi-miR-106 b-5 p）在7个胎儿阶段的皮肤样本中的表达趋势与测序结果一致。测序结果可靠。TGF-β 2与chi-miR-199 a-5 p在胎儿45 d-135 d表达的相关系数为-0.84，呈较强的负相关，初步判断二者的靶向关系。双荧光素酶载体报告结果显示，chi-miR-199 a-5 p显著降低TGF-β 2 3 ' UTR荧光素酶的表达，确定二者在特定时间存在相互关系。我们将chi-miR 199 a-5 p-FAM模拟物转染到体外培养的内蒙古绒山羊成纤维细胞中。转染后，收获细胞以提取总RNA和蛋白质。RT-qPCR和western blot检测成纤维细胞TGF-β 2 mRNA和蛋白表达水平。证实了chi-miR-199 a-5 p在成纤维细胞中在mRNA和蛋白质翻译水平上抑制TGF-β 2表达。同时，再次证明TGF-β 2基因是chi-miR 199 a-5 p的靶基因。
Inner Mongolia cashmere goats, as an important part of animal husbandry production, play an important role in animal fiber industry. In recent years, scientific research has made a lot of explorations on the molecular regulation mechanism of hair follicle cycle growth, but few studies have been reported on the development of cashmere hair in fetal period. This study was based on the completion of 21 skin samples of mRNA and miRNA sequencing in 7 fetal periods (45 days, 55 days,65 days,75 days,95 days,115 days and 135 days) of the Inner Mongolia Cashmere goat. The target genes of miRNA associated with the development of secondary hair follicles in the cashmere goats were selected through the combination analysis of mRNA and miRNA data. Then the overexpression vector was constructed and the interaction between the miRNA and the target gene was identified by Dual-Luciferase Reporter Gene System. The function and interaction relationship of chi-miR-199a-5p and TGF-beta 2 were verified by RT-qPCR and western blot at the level of the fibroblasts in Inner Mongolia Cashmere goat. It provides a theoretical basis for further study of miRNA and its target genes regulating the occurrence and development of skin hair follicles. As the result shows, the expression trends of 7 genes (BAMBI, SMAD1, LTBP1, PPP2R1B, ID4, BMP8B and PITX2) and 7 miRNA (chi-miR-17-5p, chi-miR-125b-3p, chi-miR-21-5p, chi-miR-143-5p and chi-miR-106b-5p) in the skin samples for the seven stages of the fetus were shown to be consistent with the sequencing results. the results of sequencing are reliable. The correlation coefficient of TGF-beta 2 and chi-miR-199a-5p in fetal 45d-135d expression is -0.84, showing a strong negative correlation, The target relationship was preliminarily judged. The results of double luciferase vector report showed that chi-miR-199a-5p significantly decreased the expression of luciferase in TGF-beta 2 3 ' UTR, It is determined that there is a reciprocal relationship between them at a specific time. We transfected chi-miR199a-5p-FAM mimics into fibroblasts cultured in vitro from Inner Mongolia cashmere goats. After transfection, the cells were harvested to extract total RNA and protein. The mRNA and protein expression levels of TGF-beta 2 in fibroblasts were detected by RT-qPCR and western blot. It was verified that chi-miR-199a-5p inhibited TGF-beta 2 expression at both mRNA and protein translation levels in fibroblasts. At the same time, it was again proved that the TGF-beta 2 gene is a target gene of chi-miR199a-5p.