The Apoptotic Effect Of MiR-26b-5p In Ovarian GCs By Targeting CDH2 In Rats

ObjectiveTo study the regulation of miR-26b-5p on N-cadherin(CDH2)and reveal the mechanism of miR-26b-5p in the apoptosis of ovarian granulosa cells(GCs)in rats.Methods1.Isolation,culture and identification of ovarian GCs from female SD rats:The GCs of mature ovarian follicles of normal female SD rats were extracted by mechanical separating method,then the collected GCs would be cultured for 48 hours before changing liquid for the next experiment.GCs were identified by the immunocyto-chemical staining of follicle stimulating hormone receptor(FSHR)on the cell surface.2.Target binding and binding sites of miR-26b-5p and CDH2: Three bioinformatics target gene prediction software,Targetscan7.1,PicTar and miRWalk,were used to construct plasmids containing the 3'UTR regions and plasmids containing the mutational site: The pmirGLO-CDH2 UTR-WT and pmirGLO-CDH2 UTR-MUT vectors were co-transfected with miR-26b-5p mimics and cells were treated with the dual fluorescein reporter gene assay kit,the multifunctional microplate reader was used to detect the reporter gene activity.3.The role of the regulation of miR-26b-5p on CDH2 in apoptosis of GCs in mature follicles: To further investigate the role of miR-26b-5p in the regulationof CDH2 on the apoptosis of GCs in mature follicles,we verified the regulatory effect of miR-26b-5p and CDH2 on the apoptosis of mature follicle GCs and the regulatory effect of miR-26b-5p on CDH2,respectively.1)Regulation of miR-26b-5p on GCs in mature follicles.The miR-26b-5p mimics,miR-26b-5p inhibitor and miR-26b-5p negative control(NC)were transfected into GCs of mature follicles,respectively.Real-time PCR was used to detect the expression of miR-26b-5p and the mRNA of Bcl-2 and Bax after 36 hours.After 48 hours,Annexin V / PI flow cytometry was used to detect the apoptosis rate of GCs.2)Regulatory effect of CDH2 on GCs in mature follicles.The CDH2 siRNA and CDH2 NC were transfected into GCs of normal mature follicles,respectively.After 36 hours,m RNA expression of CDH2 was detected by real time quantitative PCR.After 48 hours,apoptosis rate of GCs was detected by Annexin V / PI flow cytometry.3)The regulation of miR-26b-5p on CDH2: The expression of CDH2 mRNA was detected by real time PCR 36 hours after transfection with miR-26b-5p mimics / miR-26b-5p inhibitor and miR-26b-5p NC respectively.The expression of CDH2 proteins was detected by western blots after 72 hours.4)To study the role of miR-26b-5p in the regulation of CDH2 in the apoptosis of GCs,we constructed the eukaryotic expression vector CDH2 siRNA,and then silenced it by RNA interference technology.The expression of Bcl-2 and Bax protein was detected by western blots 72 h after transfection with miR-26b-5p inhibitor respectively.Results1.The primary GCs were isolated and extracted,and then inoculated on culture dishes.After 48 hours,the cells adherently grew in a round shape with sparse cells.After passage,the cells grew in a colony-like manner.The first generation of GCs can express FSHR,while the polygon and spindle-shapedcells expressed negatively.2.The results of three bioinformatics target genes prediction software Targetscan7.1,PicTar and miRWalk showed that CDH2 is a potential target gene of miR-26b-5p,in which the 3'UTR region of CDH2 is complementary to miR-26b-5p Binding site,initially determined the possibility of miR-26b-5p transcriptional regulation of CDH2.Through a dual luciferase reporter assay and site-directed mutagenesis experiments we found that gene CDH2 is a direct target of miR-26b-5p.3.Real-time PCR revealed that miR-26b-5p was highly expressed in GCs transfected with miR-26b-5p mimics,while Bcl-2 expression decreased and Bax expression increased.The results of flow cytometry showed that miR-26b-5p could promote the apoptosis of GCs,while miR-26b-5p inhibitor could suppress the apoptosis of GCs caused by endogenous miR-26b-5p.Besides,CDH2 mRNA expression decreased and flow cytometry found that GCs apoptosis rate increased by transfecting CDH2 siRNA alone.Under the condition of miR-26b-5p inhibitor cotransfection with CDH2 siRNA,western blots technology found miR-26b-5p could suppress Bcl-2 protein expression and promote Bax protein expression.ConclusionsThis study confirmed that gene CDH2 is the target gene of miR-26b-5p and meanwhile miR-26b-5p can promote the apoptosis of gene GCs by suppressing the expression of gene CDH2.Therefore,the molecular mechanism of miRNAs promoting the apoptosis of ovarian GCs is explored,which is a further expansion of the role of miRNA plays in the process of follicular atresia and contribution to further study about molecular regulation mechanism of follicular atresia.