Study On The MiR-23a Targeting CELF1 To Regulate Apoptosis Of Buffalo Granulosa Cells

In this study,based on the micro RNA(miRNA)high-throughput sequencing results of buffalo cumulus cells in buffalo GV/MII stage oocytes,one miR-23 a,which may be associated with the apoptosis of Granulosa Cells(GCs),was selected for study among the 24 differentially expressed miRNAs significantly upregulated in cumulus cells corresponding to oocytes in stage MII.The regulation of miR-23 a and its target gene on buffalo GCs apoptosis as well as its molecular mechanism were preliminarily investigated to provide the theoretical basis for improving the efficiency of buffalo oocytes in vitro maturation.The main results are as follows:1.Screening the predicted target gene CELF1 of miR-23 a and verifying their relationship.GO and KEGG enrichment analysis methods were performed to analyze 1237 predicted target genes of miR-23 a and found these target genes were mainly involved in the regulation of ribosome and other signaling pathways,which mainly focus on the regulation of cell proliferation,apoptosis,and substance metabolism,and the key target gene CELF1 was selected.The expression pattern of miR-23 a in buffalo different tissues was analyzed by RTq PCR,and the results showed that miR-23 a was highly expressed in ovary,but it was micro-expressed in testis and epididymis.To further verify the targeting relationship between miR-23 a and CELF1,RT-q PCR results showed that adding miR-23 a mimic in buffalo GCs significantly reduced the expression of CELF1(P<0.05).Furthermore,the result of dual-luciferase assay also proved the targeting relationship between miR-23 a and CELF1.2.The effect of miR-23 a targeting CELF1 on apoptosis of buffalo GCs was investigated.MiR-23 a mimic/inhibitor was added when buffalo GCs were cultured in vitro.Flow cytometry results showed that miR-23 a mimic could significantly promote the apoptosis of buffalo GCs(P<0.05).Ed U staining showed that miR-23 a mimic inhibited the cell proliferation(P<0.05),when miR-23 a inhibitor was used,opposite results were got.Secondly,pc-DNA3.1-CELF1 overexpression vector was constructed and transfected into buffalo GCs.The results showed that overexpression of CELF1 could significantly promote the proliferation of buffalo GCs and inhibit the apoptosis of buffalo GCs(P<0.05).The results of overexpression of CELF1 was consistent with the miR-23 a inhibitor.Moreover,the relative expression level of apoptosis-related genes and proteins were detected by RT-q PCR and western blot.miR-23 a mimic promoted the m RNA and protein expressions of apoptosis-related genes such as BAX,Caspase3 and P53 in buffalo GCs,inhibited the expression of anti-apoptotic gene Bcl2 m RNA and protein.The results of CELF1 and miR-23 a mimic were opposite.Furthermore,when both miR-23 a and CELF1 were transferred into buffalo GCs,it was found that CELF1 could reverse the apoptotic effect of miR-23 a on buffalo GCs.The effects of miR-23 a and CELF1 on steroid secretion of GCs in buffalo were detected by ELISA.The results revealed that miR-23 a mimic significantly decreased the secretion of estradiol(E2)(P<0.05),but the secretion of progesterone(P4)was significantly increased(P<0.05).The results of overexpression CELF1 were opposite to the addition of miR-23 a mimic.When both miR-23 a and CELF1 were co-transfected into buffalo GCs,CELF1 could reverse the inhibitory effect of miR-23 a on E2 secretion of GCs.In addition,to further verify the molecular mechanism of miR-23 a on the regulation of apoptosis by targeting CELF1,the expression level of related genes in PI3K/AKT pathway were detected by RT-q PCR.The results showed that miR-23 a decreased the expression level of PI3K(P<0.05)and AKT(P>0.05),and increased the expression of pathway gene PTEN(P<0.05).The co-transfer of miR-23 a and CELF1 had relieved the inhibitory effect of miR-23 a on the expression of PI3 K and AKT genes(P<0.05)and inhibited the promoting expression of miR-23 a on PTEN gene(P<0.05).These results indicated that :(1)miR-23 a was highly expressed in buffalo ovaries,and there was a targeting relationship between miR-23 a and CELF1.(2)miR-23 a promoted the apoptosis of GCs in cultured buffalo,decreased E2 secretion and promoted P4 secretion.In contrast,CELF1 promoted the proliferation of GCs and increased E2 secretion but decreased P4 secretion.By targeting CELF1,miR-23 a was able to reduce apoptosis,promote cell proliferation and E2 hormone secretion in buffalo GCs.(3)miR-23 a targeting CELF1 may regulate the apoptosis of buffalo GCs by inhibiting the PI3K/AKT signaling pathway in cells.