Study On The Target Gene Of Chi-miR-130b-5p In Ovarian Granulosa Cells Of Goats

MicroRNAs(miRNAs)are a class of noncoding small RNAs which widely exist in eukaryotic cells and play an important role in gene expression regulation.miRNA could degradate target mRNAs or inhibit the translation to up-regulate or down-regulate gene expression by complete or incomplete pairing to the 3’UTR region of the target gene mRNA.Small changes in the expression of miRNA lead to significant changes in the expression of the target gene.Studies showed that miRNA plays an important role in cell division,differentiation,migration and apoptosis.Recent studies also showed that miRNA regulates ovarian function mainly by acting on ovarian granulosa cells.Our experiment was aimed to identify the target gene of differentially expressed chi-miR-130b-5p in goat follicles found in our previous work.Our result showed as following:1.Prediction of target gene of chi-miR-130b-5p: chi-miR-130b-5p(P<0.01)was found differentially expressed in ovulatory and subordinate follicles of Dazu black goat(P<0.01)and predicted to target to BMP15 gene involved in ovarian hormone synthesis pathway through miRanda software and enrichment pathway analysis.2.Analysis of the interaction between chi-miR-130b-5p and BMP15-3’UTR: the sequence of BMP15-3’UTR was amplified by PCR and the recombinant luciferase reporter was constructed;the reporter vecter,mimics and mimics control were transfected into granulosa cells and luciferase activities were detected using luciferase assays.We found that the luciferase activity of test group was significantly down regulated compared to control groups indicating that BMP15-3’UTR was targeted by chi-miR-130b-5p(P<0.05).3.Identification of chi-miR-130b-5p targeting BMP15 in granulosa cells: the mimics and mimics control were transfected into goat ovarian granulosa cells,respectively,and the expression of BMP15 was detected using real time PCR and western blotting.The result showed that the expression of BMP15 was significantly decreased at mRNA and protein level in mimics group compared to control in ovarian granulosa cells(P<0.05).This results indicated that chi-miR-130b-5p which was significantly differentially expressed in ovulatory and subordinate follicles of Dazu Black Goat targeted to BMP15 in ovarian granulosa cells.