The Mechanism Of MiR-222 Regulates Immature Porcine Sertoi Cell Proliferation And Apoptosis Through Targeting GRB10 Gene

MicroRNAs(miRNAs)is a class of endogenous non-coding RNAs approximately 22 nt in length.The regulation of gene expression at the post-transcriptional level is widely involved in the regulation of various biological processes,and plays an important regulatory role in the process of testicular development and spermatogenesis.The miR-221/222 gene family is clustered and transcribed in a large cistron form,which can negatively regulate the expression of the target gene protein and participate in many important physiological,pathological processes and cell signal transduction pathways.Previous studies have shown that the miR-221/222 gene family is a small,non-coding RNA that is widely present in eukaryotes and highly conserved in evolution.It is differentially expressed in various stages of porcine testicular tissue development.In this study,dual luciferase reporter gene detection technology,quantitative real-time PCR technology(qRT-PCR),flow cytometry,CCK8 kit and EdU kit were comprehensively used to explore the role of miR-222 in targeting porcine immature Sertoli cell proliferation and apoptosis by targeting GRB10 gene.The results of the study are as follows:1.Five candidate coding genes(GAPDH,TBP,?-actin,SDHA and B2M)and five candidate miRNA reference genes(U6,ssc-miR-17-5p,ssc-miR-26 a,ssc-miR-27 a,and ssc-miR-103)in porcine testes of eight different developmental stages(E90,D1,D30,D60,D90,D120,D150,DM)were tested for their expression by using qRT-PCR.The analysis of the expression stability value(M)of ten candidate internal reference genes in porcine testis tissue in different periods have showed that the stability of the five candidate internal reference genes was: TBP> ?-actin> B2M> SDHA> GAPDH;the stability of five miRNA candidates was: U6> ssc-miR-27a> ssc-miR-17-5p> ssc-miR-103> ssc-miR-26 a.2.the miR-221/222 gene family members of each species were searched in the miRBase database.The position of miR-221/222 in the genome was determined by using Ensembl database.The CLustal W software was used to analyze the multiple sequence alignment analysis of the mature sequence.The miR-222 target gene was predicted by miRanda,TargetScan and miRWalk.The results showed that the miR-221/222 genes were located in the intergenic region except for a few species,and the homology of miR-221/222 was high.The high homology of miR-221/222 gene is highly conserved among different species,and predicted one potential target gene GRB10.3.A psiCHECK2-GRB10-3'UTR-WT/MUT vector was constructed and the vector was co-transfected with miR-222 mimic/negative control into porcine immature Sertoli cells for detection of cell luciferase activity.The result shows that miR-222 can target the binding site of GRB10 3'UTR.Ovrexpress/ inhibit miR-222 and interference the target gene GRB10 in porcine immature Sertoli cells to detect the expression of miR-222 and GRB10.The results showed that the expression level of miR-222 in porcine immature Sertoli cells overexpression/inhibition miR-222 expression was significantly higher than that in the negative control group(P<0.01);The expression of GRB10 in porcine immature Sertoli cells overexpressing miR-222 was significantly lower than that in NC group(P<0.05).The expression of GRB10 was significantly higher than that in NC group(P<0.01).The expression of GRB10 is significantly reduced when it was interfered.It can be seen that miR-222 targets the GRB10 3'UTR region to predict the binding site and regulates its expression in porcine immature Sertoli cells.4.After transient transfection of miR-222 mimic/miR-222 mimic NC and siGRB10/ siGRB10 NC,flow cytometry,CCK8 assay,EdU cell proliferation assay,and flow cytometry assay were used to observe cell growth,proliferation,and apoptosis.The qRT-PCR detection of apoptosis-related genes(Bax,Bcl2,Caspase-3)confirmed the biological function of miR-222 and GRB10 in cells.Flow cytometry results showed that overexpression of miR-222 and interference with GRB10 could inhibit cell proliferation and promote apoptosis.The results of CCK8 and EdU cell proliferation experiments showed that overexpression of miR-222 and interference GRB10 can inhibit cell proliferation.Flow cytometry experiments also showed that apoptosis increased significantly after overexpression of miR-222 and interference with GRB10.The qRT-PCR results of apoptosis-related genes showed that after overexpression of miR-222 and interference with GRB10,the expression of Bax and Caspase-3 which involved in the promotion of apoptosis genes were increased,and the expression of Bcl2,an apoptosis-inhibiting gene,the expression was decreased.All of the above results showed that overexpression of miR-222 and interference with GRB10 gene can inhibit the proliferation of porcine immature support cells and promote its apoptosis.The results of this experiment indicates that miR-222 may inhibit the proliferation of porcine immature Sertoli cells and promote its apoptosis through targeting GRB10 gene.