chi-miR-4110对奶山羊 Smad2基因的靶向调控作用
Regulation Function of chi-miR-4110 on Smad2 Gene in Dairy Goats

为揭示chi-miR-4110对奶山羊繁殖机能的调控作用，在前期构建的多羔（1胎3~5羔）和单羔（1胎1羔）关中奶山羊发情期卵巢差异表达miRNA文库的基础上，利用生物信息学、qRT-PCR和Western blot等技术验证chi-miR-4110的靶基因及其对靶基因的调控作用。荧光素酶检测结果表明，chi-miR-4110通过与 Smad2 基因的3′UTR相互作用导致荧光素酶活性降低，初步鉴定 Smad2为chi-miR-4110的靶基因。将chi-miR-4110 mimics和阴性对照分别转染到奶山羊卵巢颗粒细胞中，qRT-PCR和Western blotting结果表明，在奶山羊卵巢颗粒细胞中，过表达chi-miR-4110使 Smad2 mRNA以及Smad2蛋白的表达量均显著降低，表明chi-miR-4110在转录后水平对 Smad2起负调控作用。表明 Smad2是chi-miR-4110的靶基因，在奶山羊卵巢颗粒细胞中，chi-miR-4110靶向调控 Smad2的表达。
In order to explore regulation function of chi-miR-4110 in reproductive process of dairy goats,the bioinformatics analysis,qRT-PCR and western blot were used to predicate target gene of chi-miR-4110,and to verify the regulation function of chi-miR-4110 on targets based on deferentially expressed miRNA library of oestrual ovarian tissues from polytocous (3-5 kids/parity) and monotocous (1 kids/parity) Guangzhong (GZ) dairy goats.The luciferase analysis showed that chi-miR-4110 bound to the 3′ untranslated regions (UTR) of Smad2 gene and inhibited the expression of luciferase. Smad2 could be the target gene of chi-miR-4110.The chi-miR-4110 mimics and negative control were transfected into ovarian granulosa cells of dairy goat,respectively,and then the mRNA and protein expression of Smad2 were detected by qRT-PCR and western blotting.The result showed that overexpressed chi-miR-4110 significantly decreased the expression of Smad2 gene at mRNA and protein levels.It showed that chi-miR-4110 was a negative regulation factor of Smad2 gene at post transcriptional level.These results suggested that Smad2 is the target gene of chi-miR-4110 and inhibit the expression of Smad2 gene in ovarian granulosa cells of dairy goats.