Target Gene Prediction Of MiR-181a-5p And Its Role In Proliferation And Apoptosis Of Goose Granulosa Cells

MicroRNAs(miRNAs)are involved in a variety of ovarian physiological processes including steroidogenesis,cellular proliferation and apoptosis which have important regulatory effects on follicular development.Results from our previous research have shown that miR-181a-5p is abundantly present in the granulosa layer cells of geese ovarian follicles,indicating that it may have a role in regulation of granulsoa cell function.At present,studies on the function of miR-181a-5p in avian granulosa cells have not yet been reported.Therefore,in this study we firstly predicted the potential target geness and associated signaling pathways of miR-181a-5p through bioinformatical methods,and then explored the effects of miR-181a-5p on the proliferation and apoptosis of goose granulosa cells as well as its regulatory mechanisms.The main results were listed as follows:(1)Bioinformatical analysis showed that miR-181a-5p might participate in the FOXO signaling pathway to regulate the proliferation and apoptosis of granulosa cells by targeting the genes such as SIRT1,BCL2,CDKN1 B and PTEN.(2)The qPCR results showed that levels of miR-181a-5p was the highest in the 2-4mm follicles,and the lowest in the 4-6 mm follicles,followed by an increasing trend from the 4-6 mm to F5 follicles.Cluster analysis expressed that the expression pattern of miR-181a-5p and its targets changed dramaticly in the 8-10 mm and F5 follicles and it was clustered together with the cell proliferation and apoptosis-related genes(SIRT1,BCL2,CDKN1 B,PTEN).These results indicated that miR-181a-5p and its targets might be involved in the regulation of granulosa cell proliferation and apoptosis.(3)After transfection of the miR-181a-5p mimic and inhibitor of different concentrations,it was observed that the optimal transfection concentration of mimic in granulosa cells was 50 nM,and that of inhibitor was 120 nM.MTT assay showed that transfection of mimic significantly(P<0.05)decreased granulosa cell viability,but transfection of inhibitor increased cell viability(P>0.05).(4)Over-expression of miR-181a-5p resulted in an increase inthe expression of cell proliferation marker gene PCNA and several apoptosis-related genes(TP53,BAX,CASP3)but a decrease in that of anti-apoptotic gene BCL2,the opposite results were obtained afterinhibition of miR-181a-5p.These results suggested that miR-181a-5p inhibits proliferation but promotes apoptosis of granulosa cells.(5)Co-transfection of the wild-type plasmid and mimic of miR-181a-5p significantly decreased the luciferase activity(P<0.05),While its mutant-type plasmid significantly increased the luciferase activity(P<0.05).Results from the dual-luciferase reporter assay indicated that miR-181a-5p could act on the 3?UTR of SIRT1 to inhibit its mRNA expression.(6)Over-expression of miR-181a-5p significantly increased the expression of the core gene(FOXO1)and the downstream genes(CDKN1A,CDKN1B)in the FOXO signaling pathway(P<0.05)but decreased that of cell survival regulator AKT1.These results showed that miR-181a-5p might participate in the FOXO signaling pathway to induce apoptosis of granulosa cells by targeting SIRT1.In summary,miR-181a-5p could regulate the proliferation and apoptosis of goose granulosa cells through the FOXO signaling pathway by targeting SIRT1.These data may laid a foundation for further exploring the role of miR-181a-5p in goose granulosa cells.