The Study On The Mechanism Of MiR-99a Regulating The Proliferation And Apoptosis Of Porcine Granulosa Cells

MicroRNA(miRNA)is a kind of short non-coding RNA widely exist in various tissues and cells of animals with the length about 19 to 25 nt,which mediate transcription and translation and take part in regulating lots of physiological and pathological processes.MiR-99 a has a high expression in ovary while it is always downregulated in cancer tissues.The current study found that miR-99 a is related to cell apoptosis of granulosa cells in atretic follicles.However,there is less study on its regulatory role in the development of porcine follicles.This experiment is carried out to study the expression levels of miR-99 a in porcine follicles with different degrees of astresia,and to figure out its function and mechanism of regulating cell cycle,cell proliferation and apoptosis progresses.The result shows that miR-99 a holds a higher expression level in early atretic follicles than that in both healthy follicles and progress atretic follicles in porcine.The result of double luciferase reporter gene assay in HEK-293 T indicates that m TOR is the direct target gene of miR-99 a.Then the third generation of porcine granulosa cells were transfected with 50 n M miR-99 a Mimics for 48 hrs.Compared to the blank control gruop,overexpressing miR-99 a could inhibit the cell proliferation,induce in S phase retardation and accelerate the apoptosis progress of granulosa cells.Cyclin genes Cyclin D,Cyclin B are significantly downregulated(P<0.05);cyclin-dependent kinase genes CDK1,CDK4 are extreme significantly upregulated(P<0.01);the autophagy-related gene ATG13 and proapoptotic genes BAD,BAX are significantly upregulated(P<0.05);the antiapoptotic gene BCL-2 and the proapoptotic gene TP53 have no significant change(P>0.05);m TOR signaling pathway related genes m TOR is extreme significantly downregulated(P<0.01),AKT and AKT1 are significantly downregulated(P<0.05),while the protein expression level of m TOR is also obviously downregulated.After transfecting the porcine granulosa cells with miR-99 a inhibitors for 48 hrs,the cell cycle and apoptosis did not change obviously.Compared to the blank control group,the m RNA expression of CDK1,CDK4,Cyclin D,Cyclin B,BAD,BCL-2,TP53,ATG13,AKT and AKT1 have no significance(P>0.05);but BAX is significantly upregulated(P<0.05);the m RNA expression of m TOR is extreme significantly upregulated(P<0.01).But the protein expression of m TOR did not change obviously.The above results indicate that miR-99 a can inhibit cell proliferation and accelerate the apoptosis of porcine granulosa cells through targeting m TOR directly,then downregulating the expression of genes related to the AKT signaling pathway and upregulating the expression of genes related to apoptosis and autophagy.