Over-expression Of MiRNA-let-7b Regulates Apoptosis And Proliferation Of Follicular Granulosa Cells Via Targeting Map3k1 Gene

miRNA is differentially expressed in every stage of follicular development.Many miRNAs mediate reproductive endocrine functions,especially oocyte maturation and follicle formation,and regulate follicle atresia through their target genes,thereby regulating oocyte apoptosis.There are many factors affecting the apoptosis of sheep FGC.So far,it is not certain whether the let-7 family mediates apoptosis of FGC through MAP3K1.The purpose of this study was to study the mechanism and mechanism of let-7b on FGC apoptosis and development.The primary sheep FGC was collected,and different concentrations of FBS(0%,7%,10%,12%,15%,18%,20%),M199 basal medium and DMEM medium were used to culture sheep FGC in vitro,using CCK-8 kit to detect its effect on sheep FGS proliferation,to explore the optimal FBS concentration and optimal basic medium for in vitro culture of sheep FGC;then use the CCK-8 kit to detect different concentrations of FSH(0,5,10,20,30,40 IU / mL)and different concentrations of MT(0,5,10,20,30,40 ng / mL)on the proliferation of sheep FGC in vitro.Let-7b mimic let-7b inhibitor was used to transfect sheep FGC at different doses(40,60,80,100 and 120 ?M).The CCK-8 kit was used to detect its effect on sheep FGS growth and proliferation.Real-time fluorescence quantitative PCR(qRT-PCR)was used to analyze the expression level of MAP3K1 mRNA,and Western Blot method was used to analyze the expression level of MAP3K1 protein.293 T cells were co-transfected with MAP3K1-mimic-WT and MAP3K1-mimic-MU,and dual luciferase method was used to verify the targeting effect of let-7b on MAP3K1.When the FBS concentration is 12% in the two groups of M199 medium andDMEM medium,the FGC value-added rate is the highest(P <0.01),and the FGC growth is the best.As the FSH concentration is higher,the growth rate of sheep FGS increases,which promotes FGC growth.When the FSH concentration exceeds 20 IU /mL,it will have an inhibitory effect on sheep FGC in vitro culture;as the MT concentration increases,sheep FGC growth and proliferation The rate increases and promotes growth.When the MT concentration exceeds 20 ng / mL,it will have an inhibitory effect on sheep FGC in vitro.Compared with CG,the FGC proliferation activity of MIM-2,MIM-3 and MIM-4 groups was reduced.The proliferation activity of MIM-4 group was significantly lower than that of CG and MIM-1 group(P <0.05).The let-7b mimic significantly reduced the let-7b expression level of FGC.The largest reduction found was MIM-4.MIM-3 and MIM-4 groups had lower MAP3K1 mRNA and protein levels than CG.The levels of MAP3K1 mRNA and protein in MIM-3 and MIM-4 groups were lower than those in CG(P <0.05 or P <0.01).The expression levels of MAP3K1 mRNA and protein in INH-3 and INH-4 groups were higher than those in CG and INH-1 groups.Compared with GC,co-transfection of let-7b mimic significantly inhibited luciferase activity(P <0.05).The results show that the FBS concentration of 12% is the most suitable for FGC growth and development;M199 medium is more suitable for FGC in vitro culture than DMEM medium.When the FSH concentration is 20 IU / mL and the MT concentration is 20 ng / mL,FGS growth is best for sheep cultured in vitro.When the FSH concentration exceeds 20 IU / mL,or the MT concentration is greater than 20 ng/ mL,the sheep FGC in vitro Cultivation will have an inhibitory effect;let-7b can significantly inhibit the cell viability of sheep FGC and promote its apoptosis.Higher doses of let-7b mimics and inhibitors(80 ?M and 100 ?M)can significantly inhibit and promote the expression of let-7b mRNA,MAP3K1 mRNA and protein in sheepFGC.Let-7b promotes FGC apoptosis by inhibiting the MAP3K1 gene.