| CircRNAs are a class of novel endogenous non-coding RNAs(ncRNA)with well conserved sequences and widely expressed in various cells and tissues.Studies have shown that circRNAs act as " miRNAs sponges" to inhibit their activities,thereby regulating the expression of miRNAs target genes and participating in the regulation of gene expression in post-transcriptional.The establishment of endometrial receptivity is one of the key factors for embryo implantation,and the process of endometrial receptivity establishment is accompanied by the apoptosis of endometrial epithelial cells(EECs).However,the regulatory mechanism of circRNAs on EECs apoptosis is rarely studied.In this study,circRNA3890 was differentially expressed in endometrial tissues of dairy goats during the uterine receptivity(RE)or receptivity(PE)based on the high throughput sequencing data in the laboratory.Through bioinformatics prediction,it was found that circRNA3890 could be used as a "molecular sponge" to absorb miR-26b-3p,and it was speculated that circRNA3890 negatively regulated the expression of miR-26b-3p in EECs of dairy goats and regulated the expression of its target gene MDM4.The experimental subject of this study is the EECs of dairy goat,and the regulatory relationship among circRNA3890,miR-26b-3p and MDM4 in EECs was studied by RNA interference(siRNA),overexpression,stem-loop RT-qPCR,RT-qPCR,dual-luciferase activity detection system,EdU,CCK-8,WB and cell apoptosis,as well as their regulatory effects on EECs.The main results of this study are as follows:1.CircRNA3890 as ce RNA can inhibit miR-26b-3p activity in EECsRT-qPCR results showed that the expression level of circRNA3890 in PE was significantly higher than that in RE(P<0.01),however,the expression level of miR-26b-3p in RE was significantly higher than that in PE(P<0.01),which was consistent with the previous sequencing results in our research group.Moreover,miR-26b-3p and circRNA3890 were widely expressed in all kinds of tissues in dairy goat.The experimental results of the dual-luciferase activity system showed that miR-26b-3p significantly down-regulated the dual-luciferase activity of WT-circRNA3890(wild type),but had no effect on MUT-circRNA3890(mutant type)(P<0.05).Meanwhile,overexpression of circRNA3890 reduced the expression of miR-26b-3p in EECs(P<0.01),while interference with circRNA3890 promoted the expression of miR-26b-3p in EECs(P<0.01).In addition,miR-26b-3p mimic was down-regulated the expression of circRNA3890(P<0.01),while miR-26b-3p inhibitor elevated the expression of circRNA3890(P<0.01).The above experimental results showed that circRNA3890 regulated miR-26b-3p through the target site and inhibited its expression in EECs.2.CircRNA3890 and miR-26b-3p regulated the expression of MDM4 in EECsUsing bioinformatics to predict the target gene of miR-26b-3p,it was found that MDM4 may be a potential target gene of miR-26b-3p.RT-qPCR showed that the expression level of MDM4 in PE was significantly higher than that in RE(P<0.05),contrary to the expression trend of mir-26b-3p,and MDM4 was widely expressed in all tissues of dairy goats.The results of the dual-luciferase activity showed that miR-26b-3p significantly down-regulated the dual-luciferase activity of WT-MDM4(wild type)(P<0.01),and miR-26b-3p mimic down-regulated the mRNA and protein levels of MDM4 in EECs(P<0.01).In addition,overexpression of circRNA3890 was significantly up-regulated the m RNA and protein levels of MDM4 in EECs(P<0.01),interfering with circRNA3890 significantly down-regulated the m RNA and protein levels of MDM4(P<0.05,P<0.01).Meanwhile,overexpression of MDM4 could increase the expression of circRNA3890 in EECs(P<0.01),and knockdown MDM4down-regulate the expression of circRNA3890 in EECs(P<0.01).The above experimental results showed that circRNA3890 as ce RNA inhibited the activity of miR-26b-3p,and then regulated the expression of its target gene MDM4 in EECs.3.CircRNA3890,miR-26b-3p and MDM4 regulated the proliferation of EECsIn this study,CCK8 and EdU assay showed that circRNA3890 and MDM4 promoted EECs proliferation(P<0.01),while miR-26b-3p inhibited EECs proliferation(P<0.01).Meanwhile,the apoptosis test showed that circRNA3890 and MDM4 inhibited apoptosis of EECs(P<0.01),while miR-26b-3p promoted apoptosis of EECs(P<0.01).In addition,the protein expression levels of the apoptotic genes BAX,BCL2,Caspase3 and P53 were detected by Western blot(WB).The results showed that circRNA3890 and MDM4up-regulated the protein expression of BCL2 and down-regulated the protein expression of BAX,P53 and Caspase3(P<0.01).However,miR-26b-3p up-regulated the protein expression of BAX,P53 and Caspase3,and down-regulated the protein expression of BCL2(P<0.01).The above experimental results showed that circRNA3890 and MDM4 promoted EECs proliferation,while miR-26b-3p inhibited EECs proliferation.In summary,the entry point of this experiment was circRNA3890 differentially expressed in the endometrium of dairy goats in the receptivity stage and the pre-receptivity stage,which revealed circRNA3890 could be used as ce RNA to target miR-26b-3p,andremove the inhibition of target gene MDM4,thus promoting the proliferation of EECs in dairy goats.This study provided an experimental basis for further study on the molecular regulatory mechanism of receptive endometrium in dairy goats. |
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