Study on the Targeted Regulation of Wnt7a Gene Expression by miR-192 in Pigs
[Objective]The targeting relationship between procine miR-192 and Wnt family member 7A(Wnt7a)genes was identified to establish a basis for further construction of miR-192-mediated embryonic communication.[Method]The miRNA pull-down was used to identify the interactions between miR-192 and Wnt7a gene.Bioinformatics software RNAhybrid 2.2 was utilized to predict the binding sites of miR-192 and Wnt7a gene 3'-UTR.Fragments of Wnt7a gene 3'-UTR containing miR-192 binding sites were cloned into the dual luciferase reporter gene plasmid and subjected to identification through Not Ⅰ and Xho Ⅰ digestion and sequencing.The identified plasmids were co-transfected with miR-192 mimics and mimics NC into porcine endometrial epithelial cells to detect dual-luciferase activity.miR-192 mimics,mimics NC,miR-192 inhibitor,inhibitor NC were separately co-transfected with constructed Wnt7a gene 3'-UTR wild-type and mutant dual-luciferase reporter gene plasmids into porcine endometrial epithelial cells.The mRNA and protein expression of Wnt7a gene were detected by Real-time quantitative PCR and Western blotting.[Result]Pull-down results showed that the relative expression of Wnt7a gene was extremely significantly increased in miR-192 mimics group compared with its negative control group(P＜0.01),and extremely significantly decreased in the miR-192 input group compared with its negative control group(P＜0.01).The binding site(UGACCUA)between miR-192 seed region and Wnt7a gene 3'-UTR was predicted by RNAhybrid 2.2 software.The dual luciferase activity was significantly lower in miR-192 mimics group transfected with Wnt7a wild-type plasmid compared with miR-192 mimics NC group(P＜0.05),and there was no significant difference in dual luciferase activity between miR-192 mimics group transfected with Wnt7a mutant plasmid and miR-192 mimics NC group(P＞0.05).Real-time quantitative PCR results showed that the mRNA relative expression of Wnt7a gene in miR-192 mimics group was significantly decreased than that in its negative control group(P＜0.05),and miR-192 inhibitor group was significantly higher than that in its negative control group(P＜0.05).Western blotting results showed that the expression of Wnt7a protein was extremely significant inhibited after overexpression of miR-192(P＜0.01),and the expression of Wnt7a protein was extremely significant increased after inhibiting miR-192(P＜0.01).[Conclusion]miR-192 was able to target Wnt7a gene 3'-UTR and inhibit its expression.The results of this study could provide a theoretical basis for future in-depth research on the regulatory mechanism of miR-192 in the process of pregnancy implantation.
万方数据
维普
