基于microRNA-mRNA联合分析对绒山羊胚胎期次级毛囊发生发育分子调控作用的研究

On the basis of microRNA and mRNA high-throughput sequencing at different embryo stages in Inner Mongolia cashmere goat, the integrative analysis of mRNA and microRNA will be performed in this study. Combined with histological research results of hair follicles morphogenesis and development at different embryo stages of Inner Mongolia cashmere goat, the related microRNAs and their target genes will be screened. In order to confirm microRNA associated with morphogenesis and development of secondary hair follicle, the expression of microRNA in embryo skin of different stages will be detected by real time quantitative PCR.The candidate target genes will be verified by real time quantitative PCR and the luciferase reporter gene system. Mimics and inhibitors of important microRNAs will be synthesized and transfected into cultured dermal fibroblasts and epithelial cells of Inner Mongolia cashmere goat in vitro. The changes of mRNA and protein expression of the target gene and the signal pathway gene of hair follicle morphogenesis and development were detected,which could reveal molecular regulation of secondary hair follicle morphogenesis and development at embryo stages in cashmere goat. All of these will lay foundation for molecular mechanism of cashmere goat hair growth.

本研究在课题组完成绒山羊胚胎期毛囊发生发育不同阶段皮肤组织microRNA和mRNA高通量测序的基础上，对microRNA和mRNA数据进行联合信息分析，结合内蒙古绒山羊胚胎期毛囊形态发生发育变化规律的组织学研究结果，筛选与次级毛囊发生发育相关的microRNA及其作用的靶基因。通过实时定量PCR技术检测microRNA在内蒙古绒山羊胚胎期不同阶段的表达情况，确认与次级毛囊发生发育相关的microRNA。通过实时定量PCR技术和构建荧光素酶报告基因系统对重要microRNA的候选靶基因进行验证。合成重要microRNA的mimics和inhibitors，转染体外培养的内蒙古绒山羊真皮成纤维细胞、表皮细胞，检测细胞中靶基因及毛囊发生发育相关信号通路基因mRNA和蛋白质表达变化情况，揭示绒山羊胚胎期次级毛囊发生发育分子调控作用，为绒山羊绒毛生长分子调控机理奠定基础。

本研究在课题组完成绒山羊胚胎期毛囊发生发育不同阶段皮肤组织miRNA和mRNA高通量测序的基础上，对miRNA和mRNA数据进行联合信息分析，结合内蒙古绒山羊胚胎期毛囊形态发生发育变化规律的组织学研究结果，筛选与次级毛囊发生发育相关的mRNA及miRNA。通过分子生物学技术验证相关miRNA-mRNA靶向关系并探究其所在信号通路对绒山羊胚胎期次级毛囊发生发育分子调控作用，为绒山羊绒毛生长分子调控机理奠定基础。主要研究结果如下：.（1）通过对转录组数据分析得到6个比对组（45dvs55d，55dvs65d，65dvs75d，75dvs95d，95dvs115d和115dvs135d）差异表达的mRNA和miRNA。.（2）筛选出与绒山羊次级毛囊发生发育相关的mRNA共1548个。通过富集分析发现与毛囊发生发育相关的TGF-β和MAPK两条通路显著富集。TGF-β和MAPK两条信号通路中共同存在的关键基因为TGF-β2。.（3）通过WGCNA分析得到候选hub基因584个，GO和KEGG功能富集分析发现WNT10A是内蒙古绒山羊皮肤毛囊发生发育成熟阶段hub基因。.（4）利用qRT-PCR和双荧光素酶报告基因验证发现WNT10A是chi-miR-130b-3p的靶基因，TGF-βR2、FGFR2是chi-miR-370-3p的靶基因，TGF-β2是chi-miR199a-5p、chi-miR-200a的靶基因。.（5）利用qRT-PCR和Western blot检测发现在成纤维细胞中chi-miR-199a-5p,chi-miR-200a,chi-miR-200b,chi-miR-200c抑制TGF-β2表达；在成纤维细胞和上皮细胞中，chi-miR-130b-3p、chi-miR-301a-3p和chi-miR-532-3p对WNT10A有不同程度的负调控作用，chi-miR-370-3p对TGF-βR2、FGFR2均有显著的负调控作用。.（6）通过DNA染色法、CCK8法和Annexin V-FITC/PI双染色法发现WNT10A可能促进上皮细胞和成纤维细胞的增殖并抑制上皮细胞的凋亡；chi-miR-370-3p可抑制上皮细胞和成纤维细胞的细胞增殖而对细胞凋亡无显著影响。细胞划痕试验显示，chi-miR-370-3p可提高上皮细胞和真皮成纤维细胞的迁移能力。