Effects Of CircRNA211/miR-431/CSF1 Network On The Establishment Of Endometrial Receptivity In Dairy Goats

During embryo implantation,endometrial epithelial cells are remodeled and the endometrium is in the receptive phase,allowing the embryo to attach in a limited time.Therefore,the establishment of endometrial receptivity is an important prerequisite for successful embryo implantation.Abnormal endometrial receptivity of dairy goats during estrous period may lead to the failure of embryo implantation.Previous study found that circRNA211 and miR-431 were differentially expressed in endometrial receptivity through high-throughput sequencing technology.In addition,it has been reported that CSF1 is a marker gene for endometrial receptivity,but its influence on the establishment of endometrial receptivity in dairy goats is still unclear.Bioinformatics analysis showed that the circRNA211 sequence contained binding sites for miR-431.FBXO18(F-box protein,Helicase,18),the host gene of circRNA211,contained the targeting binding site for miR-431;Moreover,the CSF1 gene(colony stimulating factor 1)also had a targeted binding site for miR-431 on the 3’UTR.Therefore,this study took dairy goat endometrial epithelial cells(GEECs)as the research object,and the effects of circRNA211/miR-431/CSF1 network on endometrial epithelial cells and the establishment of endometrial receptivity of dairy goats were explored through dual luciferase reporting system,RT-q PCR,Western bolt,CCK-8,Ed U staining and flow cytometry.The main results of this study are as follows:1.miR-431 inhibited the establishment of endometrial receptivity in miceCompared with the negative control,the endometrial tissue thickness of mice injected with miR-431 agomir in the left uterine horn was significantly thinner,and the number of pinopodes on the endometrial surface was reduced.Meanwhile,vascular endothelial growth factor(VEGF)and osteopontin(OPN),marker genes of endometrial receptivity,were significantly down-regulated,and mucin antigen 1(MUC1)was significantly up-regulated(P<0.01).In addition,miR-431 significantly inhibited mouse embryo implantation.Therefore,miR-431 inhibited embryo implantation by impeding the establishment of endometrial receptivity.2.The circRNA211/miR-431/CSF1 regulatory network was found in GEECsDual-luciferase activity assay showed that circRNA211 sponged miR-431.However,miR-431 could bind CSF1 and FBXO18(P<0.01).RT-q PCR results showed that overexpression of FBXO18 significantly enhanced the expression of circRNA211(P<0.01),while interference of circRNA211 had no effect on the expression of FBXO18.In addition,both miR-431 overexpression and circRNA211 interference significantly inhibited the m RNA and protein levels of CSF1(P<0.05).The overexpression of circRNA211 and FBXO18 significantly increased the m RNA and protein levels of CSF1(P<0.05).In conclusion,FBXO18 positively promoted the expression of circRNA211,which inhibited miR-431,and miR-431 targeted CSF1 and FBXO18 down-regulation.3.circRNA211/miR-431/CSF1 network promoted the proliferation of GEECsCCK-8 and Ed U staining results showed that overexpression of FBXO18,circRNA211 and CSF1 significantly promoted the proliferation of GEECs,while co-transfection of miR-431 reduced the proliferation to a certain extent(P<0.01).In contrast,miR-431 or interference with CSF1/circRNA211 significantly inhibited cell proliferation(P<0.01).Meanwhile,flow cytometry and WB results showed that overexpression of CSF1,circRNA211 and FBXO18 significantly inhibited the apoptosis of GEECs,while miR-431 or interference with CSF1/circRNA211 significantly promoted the apoptosis of GEECs.In summary,FBXO18 positively regulated circRNA211,which acted as a ce RNA to sponge miR-431,thereby weakening the negative regulatory effect of miR-431 on target gene CSF1 and FBXO18.This study revealed the regulatory network of circRNA211/miR-431/CSF1 and its influence on endometrial epithelial cells of dairy goat,which provided an experimental basis for further exploring the regulatory mechanism of uterine receptivity of dairy goats,and a theoretical basis for improving reproductive rate of dairy goats.