绒山羊胎儿皮肤组织中miR-199a-5p的表达特征及其靶基因研究

Expression of miR-199a-5p in Fetal Skin Tissues of Cashmere Goats and Research on Its Target Genes

该研究旨在分析miR-199a-5p在陕北白绒山羊毛囊发生发育过程中的作用。采集陕北白绒山羊胚胎期65 d(E65)和120 d(E120)的皮肤组织,通过实时荧光定量PCR(qPCR)分析miR-199a-5p的表达变化趋势。利用miRBase数据库对miR-199a-5p进行保守性分析。通过TargetScan、PicTar和miRanda等生物信息学软件预测miR-199a-5p的靶基因,结合相关文献报道和课题组前期陕北白绒山羊胚胎期皮肤组织转录组测序结果进行筛选,并对这些靶基因进行GO和KEGG pathway富集分析。最终将JUNB基因作为miR-199a-5p的潜在靶标,并通过双荧光素酶试验和qPCR进行验证。qPCR结果表明,miR-199a-5p在E65(毛囊诱导阶段)高表达,E120(毛囊分化阶段)低表达,表明miR-199a-5p可能在毛囊发生发育过程中起重要作用。根据miRBase数据库相关信息可得,miR-199a-5p在各物种间非常保守。通过生物信息学、文献报道和课题组陕北白绒山羊胚胎期皮肤组织转录组测序结果联合分析共获得88个靶基因,其主要富集在细胞组分、细胞进程和大分子结合等过程,涉及调节干细胞多能性、TNF及MAPK等信号通路。结合毛囊发生发育相关文献推测,转录因子JUNB在毛囊分化阶段起重要作用。双荧光素酶试验结果表明,miR-199a-5p能够与JUNB基因的3'UTR相结合,qPCR进一步表明miR-199a-5p靶向调控JUNB并抑制其表达。miR-199a-5p可能通过调控JUNB的表达参与毛囊发生发育过程。

The purpose of this study was to analyze the role of miR-199a-5p in the development of hair follicles in Shaanbei white cashmere goats.The embryonic skin tissues of Shaanbei white cashmere goat at 65 d(E65)and 120 d(E120)were collected,and its expression was analyzed by real-time quantitative PCR(qPCR).The conservative analysis of miR-199a-5p was performed using miRBase database.The target genes of miR-199a-5p was predicted through TargetScan,PicTar and miRanda,and screened by combining relevant literature reports and our previous transcriptome sequencing results of skin tissue of embryonic Shaanbei white cashmere goats.Then,these target genes were analyzed by GO and KEGG pathway enrichments.Finally,JUNB gene was taken as the potential target of miR-199a-5p,and verified by dual luciferase assay and real-time quantitative fluorescence PCR.The results show that miR-199a-5p expressed significantly higher at E65 stage(hair follicle induction stage)than that at E120 stage(differentiation stage),suggesting that miR-199a-5p may play an important role in the development of hair follicles.According to the relevant information in the miRBase database,miR-199a-5p is very conserved among various species.A total of 88 target genes were obtained by combining bioinformatics,relevant literature reports and our previous transcriptome sequencing results of skin tissue of embryonic Shaanbei white cashmere goats,which were mainly enriched in cell components,cell processes and macro molecular binding processes,and involved in the regulation of stem cell pluripotency,TNF and MAPK signal pathways.According to the literature on the development of hair follicles,the transcription factor JUNB plays an important role in the differentiation stage of hair follicles.The dual luciferase assay indicated that the 3'UTR of JUNB had the binding site of miR-199a-5p.The qPCR result further showed that miR-199a-5p could target JUNB gene and inhibit its expression.It is speculated that miR-199a-5p may participate in the process of hair follicle development by regulating JUNB gene.