Mechanism Of MiR-222 Targeting THBS1 Inhibiting Apoptosis Of Porcine Follicular Granulosa Cells

During the growth and development of sow follicles,more than 99%of follicles selectively disappear,called "locking",and only a few follicles ovulate during follicular development.The total number of ovulations is regulated by genes,called "ovulation rate",which is an important parameter for the reproductive efficiency of farm animals,especially sows.Ovulation rate is the main limiting factor in determining the number of offspring.Apoptosis of granulosa cells affects follicular atresia and reproduction.The mechanism of granulosa cell apoptosis may be the key to follicular atresia research and is regulated by the expression of miRNA and certain genes.Regulatory pathways involved in resveratrol include inhibition of cell proliferation by inhibition of cell proliferation pathways and cyclin-dependent kinases(cdks),promotion of cell differentiation,induction of apoptotic cell death by activation of mitochondria-dependent or independent pathways,etc.Its research on porcine ovarian granulosa cells is still not perfect.Apoptosis of granulosa cells is not only regulated by miRNAs,but also by certain genes.For example,various animal models have been used to study the role of THBS1 in follicular function or atresia,but the biological function of THBS1 in pGCs and its correlation The regulatory mechanism is still unclear.In this study,we used porcine follicular granulosa cells as the research object.Firstly,we analyzed the function of resveratrol on porcine ovarian granulosa cells,including its effects on porcine follicular granulosa cell morphology,proliferation,cell cycle and hormones.The regulatory relationship between resveratrol and miR-222.On this basis,the effects of miR-222 on apoptosis of porcine ovarian granulosa cells and its mechanism of action were further explored.This paper has carried out the following experiments:(1)The potential biological functions of resveratrol on pGCs and the regulation of miR-222;(2)Analysis of the potential biological functions of miR-222 on pGCs and the molecular regulation mechanism of pGCs apoptosis.(3)Interfering with the expression of the THBS1 gene on the potential biological functions of pGCs and the molecular regulation mechanism of apoptosis of pGCs.(4)Analysis of the regulation mechanism of miR-222 on the expression of THBS1 gene.The above studies mainly obtained the following results:(1)Treatment of pGCs with different concentrations(0?mol/L,10 ?mol/L,25 ?mol/L,50 ?mol/L,75 p?mol/L,100 p?mol/L)with resveratrol,with increasing concentration of resveratrol The morphology of pGCs was found to change at concentrations up to 100?mol/L;proliferation of pGCs was inhibited by resveratrol and inhibited in a concentration-dependent manner;however,estrogen levels in pGCs did not change significantly.After treatment of GCs with different concentrations of resveratrol,the percentage of cells in GO/G1 phase increased,the percentage of cells in S phase and G2/M phase decreased,and the number of cells arrested in G0/G1 phase was the highest.At the same time,it was found that with the increase of resveratrol concentration,the mRNA levels of the cycle-related genes CyclinB and CyclinD decreased in a dose-dependent manner;the mRNA level of p21 was up-regulated as a whole.In addition,resveratrol can regulate the expression of miR-222,and it will decrease first and then increase with the increase of resveratrol concentration;miR-222 inhibits the apoptosis of pGCs induced by resveratrol at 75 ?mol/L Die.(2)miR-222 mimics can promote the increase of cell number and inhibit the apoptosis of pGCs;increase the expression level of anti-apoptotic BCL-2 gene and decrease the expression level of pro-apoptotic caspase-3 gene;increase the level of estrogen in pGCs.miR-222 inhibitors inhibit the increase in the number of granulosa cells and promote granulosa cell apoptosis;reducing the expression level of BCL-2.(3)After transfection of THBS1-siRNA GCs for 48h,the effect of interfering with THBS1 gene on apoptosis of GCs was detected by flow cytometry.It was found that the apoptosis rate of pGCs transfected with THBS1-siRNA was significantly lower than that of pGCs transfected with NC mimics.RT-PCR results showed that the interference with the THBS1 gene reduced the expression level of the proapoptotic gene caspase-3,while the expression level of the anti-apoptotic gene BCL-2 was not significantly affected.However,estrogen levels in pGCs were down-regulated after interference with the THBS1 gene;pGC cell viability was reduced.Interference with the THBS1 gene inhibited apoptosis of pGCs induced by resveratrol at a concentration of 75 ?mol/L.(4)miR-222 can bind to the 3'UTR region specific site of THBS1 gene,and regulate the expression of THBS1 mRNA and protein.THBS1,as a target gene of miR-222,has a direct effect on the regulation of pGCs apoptosis by miR-222.