Study Of MiR-486 On The Proliferation And Apoptosis Of Goat Ovarian Granulosa Cells By Targeting SRSF3

Micro RNAs are a class of non-coding RNAs that are widely expressed in organisms.They are involved in regulating cell proliferation,apoptosis,cycles and other processes.Previous laboratory studies found that mi R-486 significantly differentially expressed in dairy goat ovaries during lambed singleton and fecundity by high-throughput sequencing.Bioinformatics analysis indicated that mi R-486 might be involved in regulating the growth and development of ovarian follicles in dairy goats,which affects the process of ovarian follicular atresia.The folliclular atresia is accompanied by the apoptosis of granulosa cells.However,the molecular mechanism by which mi R-486 regulates granulosa cell apoptosis in dairy goats remains unknown.In this study,Guanzhong dairy goats were used as research object,and serine and arginine-rich splicing factor 3（SRSF3）was screened as a target gene of mi R-486.Whereafter,SRSF3 overexpression vector（pc-SRSF3）was constructed and interfering RNA for SRSF3（si-SRSF3）was synthesized to study the effect of SRSF3 on granulosa cells.By using CCK-8,Ed U assay,Flow cytometry assay,RT-q PCR,Western blot methods,these experiments were mainly to study the regulatory effects of mi R-486 and target gene SRSF3 on granulose cell proliferation and apoptosis of dairy goats.These mainly results were following,1. mi R-486 promotes apoptosis and inhibits proliferation of ovarian granulosa cells in dairy goatsRT-qPCR was used to detect the expression of mi R-486 in ovary,breast,uterus,pituitary,kidney,heart,lung,liver and intestine of dairy goats.The result showed that mi R-486 expressed in all tissues with the highest expression in the lung and a higher expression in the ovary.Then,mimic NC,mi R-486 mimic,inhibitor NC and mi R-486inhibitor was respectively synthesized and transfected into granulosa cells.CCK8,Ed U,and flow cytometry were used to detect the regulation of mi R-486 on granulosa cells.The results showed that mi R-486 inhibited cell proliferation and promoted cell apoptosis of granulosa cells.Subsequently,RT-q PCR and Western blot results showed that mi R-486 significantly inhibited the expression of PI3K total protein,AKT and m TOR phosphorylated proteins.On the contrary,mi R-486 significantly promoted the m RNA and protein expression of BAX,P53,Caspase-3.These results suggested mi R-486 might regulate cell proliferation through the PI3K/AKT/m TOR pathway,activate the expression of BAX,BCL-2,P53,Caspase-3 and thus promote cell apoptosis in granulosa cells.2. mi R-486 regulates the expression of SRSF3Targetscan predicted that the 3’UTR region of SRSF3 contains mi R-486 target sites.Dual luciferase reporter vector psi CHECKTM-SRSF3-WT and psi CHECK^TM-SRSF3-MU containing XhoⅠand NotⅠrestriction sites were constructed.Then the target relationship between mi R-486 and SRSF3 was verified in HEK293T cells.The results showed that mi R-486 significantly inhibited the luciferase activity of psi CHECK^TM-SRSF3-WT,while the luciferase activity of psi CHECKTM-SRSF3-MU changed indistinctive.In addition,RT-q PCR and Western blot showed that mi R-486 negatively regulated m RNA and protein levels of SRSF3 in granulosa cells.These results suggest that mi R-486 acted on the 3’UTR region of SRSF3 and inhibited the expression of SRSF3 m RNA and protein.3. SRSF3 inhibits apoptosis and promotes proliferation of ovarian granulosa cells in dairy goatsSRSF3 expressed in ovary,breast,uterus,pituitary,kidney,heart,lung,liver and intestine tissues of dairy goats.The expression in the uterus was the highest,followed by that in the ovary,and the expression in the heart,lung,liver,and intestine was lower.According to the NCBI database,the CDS length of the SRSF3 gene in goat is predicted to be 495bp.A vector pc-SRSF3 containing the full-length SRSF3 CDS sequence between Bam HⅠand Eco RⅠdigestion sites was constructed,and the interfering RNA of SRSF3（si-SRSF3）was synthesized.RT-q PCR and Western blot results showed that pc-SRSF3 significantly increased the expression of SRSF3,while si-SRSF3 significantly decreased the expression of SRSF3 in granulosa cells.In addition,CCK8,Ed U,and flow cytometry results showed that SRSF3 overexpression significantly inhibited cell apoptosis,promoted cell viability and proliferation,while SRSF3 depletion promoted cell apoptosis,and inhibited cell viability and proliferation.Besides,SRSF3 overexpresson significantly inhibited the expression of BAX,P53,Caspase-3,promoted the BCL-2 expression.SRSF3 depletion showed the opposite results.4. Regulation of mi R-486 on ovarian granulosa cell proliferation and apoptosis by targeting SRSF3 in dairy goatsAfter co-transfection of mi R-486 mimic and pc-SRSF3 into granulosa cells,the effects on granulosa cells were detected by CCK8,Ed U,and flow cytometry.The results showed that compared with mi R-486 mimic,the co-transfection group significantly restored the inhibitory effects of mi R-486 mimic on granulosa cell viability and increased the number of Ed U-positive cells.SRSF3 partly neutralized the effect of mi R-486 on promoting cell apoptosis.Co-transfection group induced BAX,Caspase-3 protein expression,increased the protein expression of BCL-2 and BCL-2/BAX expression ratio significantly compared with mi R-486 mimic group,without P53 expression.Meanwhile,mi R-486 inhibitor and si-SRSF3 were co-transfected into granulosa cells.The results were consistent with the mi R-486 mimic and pc-SRSF3 co-transfection groups.However,compared with mi R-486inhibitor,the co-transfection group of mi R-486 inhibitor and si-SRSF3 significantly increased the expression of P53.Taken together,mi R-486 promotes cell apoptosis,inhibits cell viability and proliferation by negatively regulating the expression of SRSF3 in goat granulosa cells.The results provide a new experimental evidence for further exploring the molecular mechanism of mi R-486 in follicular development and atresia of dairy goats.