| Embryo implantation plays a decisive role in the conception of female livestock.The two indispensable factors of successful embryo implantation are embryo quality and endometrial receptivity.Related research showed that more than 60% of the embryo implantation failure of in vitro fertilization is caused by the endometrium receptivity abnormality,which causes heavy losses to animal production.The preliminary study of our research group showed that mi RNAs and circ RNAs may regulate the establishment of endometrial receptivity in dairy goats.However,the regulatory mechanism of mi RNAs and circ RNAs in the process of endometrial receptivity in dairy goats need to be further studied.This study used difference expression of microRNAs by early high-throughput sequencing technology in receptive(RE,D15)and pre-receptive(PE,D5)endometrium of dairy goats.Because ESCs mainly play the role of proliferation in the endometrial receptivity stage,so dual luciferase gene report system,RNA interference,overexpression vectors,immunofluorescence and other techniques were used to investigate the regulatory effects of circ RNA,mi RNA and m RNA on endometrial stromal cells and the establishment of receptive endometrium during embryo implantation of dairy goats.The results were as follows:1.The regulatory role of mi R-100-5p on ESCs proliferation The expression of mi R-100-5p in RE was significantly down-regulated compared by PE(p<0.05).In vitro,after 48 hours of treatment with mi R-100-5p mimics,the cell vitality of ESCs was reduced and the cell proliferation of ESCs inhibited.The protein expression of caspase3 was significantly increased,but the ratio of Bcl2/Bax was significantly decreased after treatment with mi R-100-5p mimics(p<0.01).In contrast,48 hours after treatment with mi R-100-5p inhibitors promoted the cell vitality of ESCs and promoted the cell proliferation of ESCs.The protein expression of caspase3 was significantly down-regulated,while the ratio of Bcl2/Bax was significantly up-regulated after treatment with mi R-100-5p inhibitors(p<0.01).2.Regulation of HOXA1 on ESCs proliferation,the target gene of miR-100-5p,In this study,by constructing the dual lucifase reporter,the results showed that HOXA1 was the target gene of mi R-100-5p,and mi R-100-5p down-regulated the expression of HOXA1 m RNA(p<0.05)and protein in ESCs(p<0.01).In addition,the expression of HOXA1 in RE was significantly increased than that in PE(p<0.05).Besides,we found that HOXA1 X1 was missing,and HOXA1 X2 was a perfect match with the predicted fragment during the construction of HOXA1 overexpression vector.In vitro,over-expressed HOXA1 promoted the cell vitality of ESCs and promoted the cell proliferation of ESCs.The protein expression of caspase3 was significantly down-regulated,while the ratio of Bcl2/Bax was significantly up-regulated after treatment with overexpression of HOXA1(p<0.01).Conversely,HOXA1 si RNA reduced the cell vitality of ESCs and inhibited the cell proliferation of ESCs.The protein expression of caspase3 was significantly increased,but the ratio of Bcl2/Bax was significantly decreased after treatment with knockdown of HOXA1(p<0.01).3.Circ-9110 acts as ce RNA to regulate ESCs proliferation through sponging mi R-100-5p In this study,by constructing a dual luciferase reporter,the result showed that circ-9110 sponged mi R-100-5p,and circ-9110 down-regulated the expression level of mi R-100-5p in ESCs(p<0.05).In addition,the expression of circ-9110 in RE was significantly higher than that in PE(p<0.05).In vitro,circ-9110 promoted the cell vitality of ESCs and promoted the cell proliferation of ESCs.The protein expression of caspase3 was significantly down-regulated,while the ratio of Bcl2/Bax was significantly up-regulated after treatment with overexpression of circ-9110(p<0.01).Conversely,circ-9110 si RNA reduced the cell vitality of ESCs and inhibited the cell proliferation of ESCs.The protein expression of caspase-3 was significantly increased,but the ratio of Bcl2/Bax was significantly decreased after treatment with knockdown of circ-9110(p<0.01).4.Molecular regulation of circ-9110/ miR-100-5p /HOXA1 on ESCs proliferation In ESCs,mi R-100-5p reduced the relative protein expression levels of p-PI3 K,p-AKT,p-m TOR and p-ERK1/2,thereby inhibiting the PI3K/AKT/m TOR and REK1/2 pathways(p<0.01),while HOXA1 and circ-9110 activated the PI3K/AKT/m TOR and ERK1/2 pathways.Circ-9110 can be used as ce RNA to sponge mi R-100-5p and then regulate ESCs.In summary,circ-9110,mi R-100-5p and HOXA1 were differentially expressed in the receptive endometrium of dairy goats.It was preliminarily revealed that circ-9110 as ce RNA synergizing mi R-100-5p involved in the regulation of the receptive endometrium of dairy goats from the three levels of circ RNA,mi RNA and m RNA.This study provides experimental basis for establishing endometrial receptivity and improving the reproductive performance of dairy goats. |
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