| Mammalian follicular development is a complex physiological process regulated by multiple factors such as endocrine,autocrine and paracrine.In-depth study of the intrinsic molecular mechanism of porcine follicle development is of great significance for the genetic improvement of sow litter size.In our previous study,390 mi RNAs were differentially expressed between the pre-ovulatory follicles of Large White(LW)and Chinese Taihu(CT)sows.Among them,mi R-17-5p was significantly differently expressed between the two pig breeds.In this study,dual luciferase reporter system,FCM(Flow Cyto Metry),CCK-8,ELISA,q RT-PCR and Western blot were used to explore the function and molecular regulation mechanism of mi R-17-5p and E2F1(E2F transcription factor 1)gene in porcine follicular development.The main research results are as follows:1.Identification of the targeting relationship between mi R-17-5p and E2F1 genes in porcine ovarian granulosa cellsThe predicted results of Target Scan software indicated that the 3'UTR region of E2F1 gene contained two potential binding sites(387-393 nt;980-986 nt)for mi R-17-5p.The double luciferase activity assay showed that mi R-17-5p inhibited the luciferase activity of E2F1 by binding to the first potential binding site(387-393 nt)of the E2F1 3'UTR.The results of q RT-PCR and Western blot indicated that mi R-17-5p regulated E2F1 expression by inhibiting the translation of E2F1 gene.2.mi R-17-5p affected the growth of porcine ovarian granulosa cellsThe results of Flow Cytometry showed that mi R-17-5p mimics down-regulated the proportion of G0/G1 phase cells and up-regulated the proportion of S phase cells;mi R-17-5p inhibitor down-regulated the proportion of S phase cells.The results of q RT-PCR showed that mi R-17-5p mimics promoted the expression of cell cycle-related gene CDK4,mi R-17-5p inhibitor had no significant effect on the expression of CDK4.The results of CCK-8 showed that mi R-17-5p mimics increased the viability of porcine ovarian granulosa cells,and mi R-17-5p inhibitor reduced the viability of porcine ovarian granulosa cells.The results of q RT-PCR showed that mi R-17-5p mimics promoted the expression of proliferation marker gene PCNA,and mi R-17-5p inhibitor suppressed the expression of PCNA.The results of Flow Cytometry showed that mi R-17-5p mimics down-regulated the proportion of porcine ovarian granulosa cell apoptosis,and mi R-17-5p inhibitor up-regulated the proportion of porcine ovarian granulosa cell apoptosis.The results of q RT-PCR showed that mi R-17-5p mimics suppressed the expression of apoptosis marker gene BAX,and mi R-17-5p inhibitor promoted the expression of BAX.3.mi R-17-5p up-regulated follicular development key genes expression and estrogen synthesisThe results of q RT-PCR and Western blot showed that mi R-17-5p mimics promoted the expression of follicular development key genes LHR,CYP19A1 and AREG,and mi R-17-5p inhibitor suppressed the expression of LHR,CYP19A1 and AREG.The results of ELISA showed that mi R-17-5p mimics up-regulated the concentration of estrogen,and mi R-17-5p inhibitor down-regulated the concentration of estrogen.4.Inhibition of E2F1 gene expression affected ovarian granulosa cell growthRNAi was used to inhibit E2F1 gene expression.The results of Flow Cytometry showed that E2F1 si RNA down-regulated the proportion of G0/G1 phase cells and up-regulated the ratio of S phase and G2/M phase cells.The results of CCK-8 showed that E2F1 si RNA increased the viability of porcine ovarian granulosa cells.The results of Flow Cytometry showed that E2F1 si RNA down-regulated the proportion of porcine ovarian granulosa cell apoptosis.5.Inhibition of E2F1 gene expression up-regulated follicular development key genes expression and estrogen synthesisThe results of q RT-PCR and Western blot showed that E2F1 si RNA promoted the expression of LHR,CYP19A1 and AREG.The results of ELISA indicated that E2F1 si RNA up-regulated the concentration of estrogen.6.Inhibition of E2F1 expression up-regulated the activity of CYP19A1 promoterThe double luciferase activity assay showed that E2F1 si RNA up-regulated the luciferase activity of the CYP19A1 promoter.The results of Ch IP assay showed that E2F1 didn't bind to the potential binding site in the promoter region of CYP19A1 gene.In conclusion:(1)mi R-17-5p promotes the proliferation of porcine ovarian granulosa cells and inhibits the apoptosis of porcine ovarian granulosa cells by directly targeting the E2F1 gene.(2)mi R-17-5p promotes the expression of follicular development key genes LHR,CYP19A1,AREG and the synthesis of estrogen by directly targeting the E2F1 gene in porcine ovarian granulosa cells. |
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