Study On The Regulation Of MiR-196a/RCC2 On The Proliferation And Apoptosis Of Porcine Immature Sertoli Cells In Vitro

microRNAs(miRNAs)are single-stranded,non-coding small RNAs composed of about 22-27 nucleotides that are widely found in eukaryotic cells.miRNAs are complementary base pairing with the mRNA of target genes,leading to degradation or translation inhibition of mRNA and regulating gene expression at the post-transcriptional level.The regulatory network is composed of miRNA and its target genes is involved in a variety of biological processes including cell proliferation,cell differentiation,cell apoptosis and development.The relative stability of their number in testis and normal physiological function play a crucial role in maintaining the testicular function and sperm production quality of male animals.Previous studies based on Solexa deep sequencing technology found that the expression of miR-196 a in immature testicles of swine was significantly higher than that in mature testicles,indicating that miR-196 a may have an impact on testicular development and spermatogenesis.Immature Sertoli cells have secretion function and strong ability of proliferation.The study aims to explore the expression,function and action mode of miR-196 a in immature Sertoli cells of swine,thus providing a research basis for testicular development and spermatogenesis of swine.To explore the effect of miR-196 a on the proliferation and apoptosis of immature Sertoli cells of porcine testis in vitro,firstly,miR-196 a mimics vector(miR-196 a mimics),inhibitor vector(miR-196 a inhibitor)and control vector(microRNA-small hairpin negative control,miRNA-ShNC)were transfected into cultured supporting cells.Cell activity was detected by MTS,cell cycle and apoptosis were detected by flow cytometry,and the expression level of Caspase 3 was detected by RT-qPCR.The results showed that the cell activity was higher and the apoptosis ratio was lower in miR-196 a mimics group than that in the control group,while the cell activity decreased and the apoptosis rate increased in the miR-196 a inhibitor group,indicating that miR-196 a promote the proliferation and inhibit apoptosis of Sertoli cells in vitro.Secondly,the binding relationship between miR-196 a and the screened target genes was detected by the dual-luciferase reporter gene detection system,and the regulatory effect of miR-196 a on the mRNA and protein expression levels of the target genes was verified by RT-qPCR and Western Blot.The miR-196 a sequence was searched through the miRBase website,and it is found that miR-196 a might bind to the mRNA of RCC2 through TargetScan database analysis.The surrounding sequence containing the-ACTACCT-sequence in the 3'UTR of the RCC2 gene was inserted into the dual luciferase gene reporter vector to obtain the wild type vector(RCC2-WT).The-ACTACCT-sequence was replaced by the-CATGTAGsequence and inserted into the dual luciferase gene reporter vector to form the mutant vector(RCC2-mut).The RCC2-WT,RCC2-mut and psiCHECK-2 vectors were respectively co-transfected with miR-196 a mimics to the Sertoli cells,and the fluorescence activity was detected 36 hours later.The results showed that the fluorescence activity of the RCC2-mut group and the psiCHECK-2 group had no significantly difference,and both of them were higher than that of the RCC2-WT group.After transfecting of miR-196 a mimics/ inhibitor and miRNA-ShNC to the Sertoli cells,RCC2 expression was detected by RT-qPCR and Western Blot.The results showed that the expression of RCC2 decreased in the miR-196 a mimics group and increased in the miR-196 a inhibitor group compared with the control group,indicating that RCC2 was a target gene of miR-196 a in the Sertoli cells.Finally,the study investigated whether miR-196 a had an effect on cell proliferation and apoptosis through RCC2.The candidate interfering fragments(RCC2 siRNA-1,siRNA-2 and siRNA-3)and Negative Control(siRNA Control)were transfected into Sertoli cells.The expression of RCC2 was detected by RT-qPCR and Western Blot.The interference effect of RCC2 siRNA-1 was the best.Taking miR-196 a inhibitor co-transfected with RCC2 siRNA-1 as the experimental group,and miR-196 a inhibitor as the control group,the study detected the cell activity,cell cycle,cell apoptosis and the expression of Caspase 3 in each group.The results showed that the cell activity of the experimental group was higher than that of the control group,the cell number of G0/G1 phase was lower than that of the control group,and the content of G2 phase cells was higher than that of the control group.There was no significant difference in the percentage of apoptotic cells and the expression of Caspase 3 between the experimental group and the control group.It can be speculated that RCC2 alleviate the proliferation inhibition caused by miR-196 a to some extent,but it had no significant effect on cell apoptosis.In conclusion,miR-196 a can inhibit the apoptosis of Sertoli cells in vitro and promote the proliferation of Sertoli cells in vitro by down-regulating the expression of RCC2.