The Study Of Molecular Regulatory Mechanism CircRNA3669 As CeRNA On Endometrial Epithelial Cells Apoptosis In Dairy Goats

Circular RNA(CircRNA)is a class of endogenous non-coding RNA with ceRNA(competitive endogenous RNA)function.Previous studies of our research group have shown that miR-26 a promoted the proliferation of endometrial epithelial cells(EEC)in dairy goats,but its molecular regulation mechanism is still unclear.According to the circRNA library constructed by our previous study,circRNA3669 was found specifically expressed in the pre-receptive endometrium of dairy goats.In addition,it was predicted that circRNA3669 could act as a ceRNA by sponging miR-26 a though the bioinformatics analysis.Therefore,this study speculated that circRNA3669 could function as a ceRNA to regulate the expression of relevant target genes through miR-26 a,thereby regulating EEC proliferation and apoptosis.In this study,EEC was used as the research object.The regulation of circRNA3669,mir-26 a and RCN2 in the EEC was studied by using interference,over-expression,stem loop RT-qPCR,RT-qPCR,double luciferase activity detection system,immunohistochemistry,Western blot,CCK-8,EdU,and cell apoptosis detection technologies.As well as the regulatory effect of circRNA3669 and RCN2 on EEC,MAPK signaling pathway and some related apoptotic proteins such as BCL2,BAX and Caspase3.The main results are as follows:1.circRNA3669 could act as ceRNA to sponge miR-26 a and inhibit the activity of miR-26aThe study found that circRNA3669 was significantly down-regulated in the receptiveendometrium of dairy goats compared to pre-receptive endometrium.The dual-luciferase assay showed that miR-26 a significantly down-regulated the dual-luciferase activity of WT-circRNA3669,so miR-26 a could be sponged by circRNA3669.In EEC,circRNA3669 significantly down-regulated the expression level of miR-26 a,but the expression level of circRNA3669 was significantly up-regulated by miR-26 a inhibitor.These results suggested that circRNA3669 could act as ceRNA to sponge miR-26 a and inhibit the activity of miR-26 a in EEC.2.circRNA3669 regulated the expression level of RCN2 by sponging miR-26aThe assay results of dual-luciferase activity showed that miR-26 a significantly down-regulated the dual-luciferase activity of WT-RCN2-3’UTR,suggesting that RCN2 was a target gene of miR-26 a.Meanwhile,miR-26 a inhibitor significantly up-regulated the RCN2 mRNA and protein levels in EEC,and miR-26 a significantly down-regulated the of RCN2 protein level.These results furtherly indicated that miR-26 a down-regulated the expression level of RCN2 in EEC by the target site.The expression level of RCN2 was significantly down-regulated in receptive endometrium than that in pre-receptive endometrium of dairy goats.In addition,circRNA3669 significantly up-regulated the RCN2 protein level in EEC,and sicircRNA3669 obviously down-regulated the level.These results suggested that circRNA3669 could function as ceRNA to up-regulate the expression level of RCN2 by sponging miR-26 a in EEC.3.CircRNA3669 and RCN2 inhibited EEC apoptosis by regulating MAPK signaling pathwayIn this study,the effects of circRNA3669 and RCN2 on the proliferation and apoptosis of EEC were detected by EdU,CCK-8,and Western blot.The results of EdU and CCK-8 assay showed that circRNA3669 and RCN2 significantly promoted EEC proliferation,but siRCN2 significantly inhibited EEC proliferation.In addition,the results of cell apoptosis analysis showed that circRNA3669 and RCN2 both obviously inhibited EEC apoptosis.Meanwhile Western blot results showed that circRNA3669 and RCN2 both up-regulated protein the levels of BCL2 and p-ERK/ERK,and down-regulated the protein levels of BAX,Caspase3,p-JNK/JNK,and p-P38/P38 in EEC.All above results suggested that circRNA3669 and RCN2 could inhibite EEC apoptosis by regulating MAPK signaling pathway.In summary,this study investigated the differential expression of circRNA3669 in receptive and pre-receptive endometrium of dairy goats,and revealed the mechanism of circRNA3669 acts as a ceRNA to regulate RCN2 by binding miR-26 a in EEC.Therefore,this study would provide experimental basis for further study on the endometrial epithelial cells of dairy goats.