Screening Embryo Implantation Related Circular RNA In Early Pregnant Mice Endometrium And Their Potential Functions

Object:Embryo implantation is one of key steps in reproduction.To explore its mechanism will help us solve many problems,such as diagnosis and treatment of infertility,improving the success rate of assisted fertility and seeking new regulation methods for reproduction.Circular RNA(circRNA)is a class of newly discovered endogenous non-coding RNA,and is structured as a covalent closed continuous loop.As a type of competing endogenous RNA(ce RNA),circRNA has been found to play important roles in regulating gene expression by sponging micro RNA(miRNA)and participate in several biological and pathological processes.However,its specific roles in embryo implantation remain unclear.This study aims to investigate the roles of circRNA in embryo implantation.Method:1.circRNA microarray assay and verification(1)Screen expression profiles of circRNA between implantation sites and inter implantation sites at day 5(D5)pregnant mice endometrial tissues using Arraystar circRNA microarray assay.(2)RT-qPCR of 4 dysregulated circRNA were performed to validate the circRNA microarray results.2.Bioinformatics assay of differentially expressed circRNA(1)Based on circRNA microarray data in current research and RNA-seq data obtained by our group previously,differentially expressed circRNA,miRNA and mRNA were used to conjoint analysis to construct circRNA—miRNA—mRNA negative correlation network.(2)The host genes of differentially expressed circRNA were used for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.(3)The target genes in circRNA—miRNA—mRNA negative correlation networks were used for GO and KEGG pathway analysis.3.The role of circFoxo3 in endometrium decidualization of early pregnant mice(1)The early pregnant mice model,artificially induced decidualization mice model and endometrial stromal cells in vitro decidualization model were established,and RT-qPCR was used to detect the expression of circFoxo3.(2)The function of circFoxo3 in endometrial stromal cells decidualizationStromal cells were up-regulated circFoxo3 expression by transfection with circFoxo3 overexpression vector,then the expression of decidualization marker Hoxa10 was detected by RT-qPCR and cellular morphology was observed by F-actin staining after in vitro decidualization.(3)Prediction and verification target miRNA-mRNA of circFoxo3Target miRNA of circFoxo3 was predicted by Target Scan and miRanda databases.Target mRNA of miR-138-5p predicted by Micro T,Pictar,miRmap and Target Scan databases,analyzed jointly with differentially down-regulated mRNA obtained fromRNA-seq data performed by our group previously,and we got potential target mRNA of miR-138-5p.Luciferase reporter analysis was used to verify their binding relationship.(4)Stromal cells were up-regulated circFoxo3 expression by transfected with circFoxo3 overexpression vector,then RT-qPCR and western blot were used to detect miR-138-5p and Klf11 protein expression after in vitro decidualization.(5)Stromal cells were up-regulated miR-138-5p expression by transfected with miR-138-5p mimic,then western blot was used to detect Klf11 protein expression after in vitro decidualization.(6)The function of Klf11 in endometrial stromal cells decidualizationThe endometrial stromal cell in vitro decidualization model was established,and western blot was used to detect the expression of Klf11 protein.Stromal cells were up-regulated Klf11 expression by transfection with Klf11 overexpression vector,then the expression of decidualization marker Hoxa10 was detected by RT-qPCR and cellular morphology was observed by F-actin staining after in vitro decidualization.Result:1.circRNA microarray assay and verification.(1)The microarray results showed 176 differentially expressed circRNA,including 101 up-and 75 down-regulated circRNA(FC?1.5 and P <0.05)at implantation sites compared with inter implantation sites.(2)RT-qPCR results showed that the expression of circRNA?008885and circRNA?013924 were up-regulated at implantation sites,whereas circRNA?21887 and circRNA?004122 were down-regulated at implantation sites.RT-qPCR results were highly consistent with the microarray analysis results,which demonstrated the high reliability of our microarray expression results.2.Bioinformatics assay of differentially expressed circRNA(1)circRNA—miRNA—mRNA negative correlation networks were constructed.21 down-regulated circRNA(circRNA?22444,circRNA?19529,circRNA?27282,etc.),14 up-regulated miRNA(miR-17-5p,miR-20a-5p,miR-361-5p,etc.)and 79 down-regulated mRNA(actr8,Lad1,sv2 b,etc.)were involved in these networks.(2)GO and KEGG analysis of the host genes of differentially expressed circRNA revealed some meaningful terms.,and the top 3 GO processes included phosphorylation,histone H4 deacetylation and histone H3 deacetylation in Biological process(BP);protein binding,nucleotide binding and ATP binding in Molecular function(MF);and cytoplasm,nucleus and replication fork in Cellular component(CC).Furthermore,KEGG pathways were identified.The top 3 pathways included endometrial cancer,thyroid hormone signaling pathway and arrhythmogenic right ventricular cardiomyopathy.(3)GO and KEGG analysis of target genes in circRNA—miRNA—mRNA negative correlation networks revealed some meaningful terms.The top 3 GO terms included activation of protein kinase B activity,positive regulation of release of cytochrome c from mitochondria and response to high density lipoprotein particle in BP;transmembrane receptor protein tyrosine kinase activity,phosphatidylinositol 3-kinase binding and phospholipid transporter activity in MF;and integral component of membrane,membrane and intracellular membrane-bounded organelle in CC.The top 3 pathways included the Rap1 signaling pathway,aldosterone-regulated sodium reabsorption and the PI3K-Akt signaling pathway.3.The role of circFoxo3 in endometrium decidualization of early pregnant mice(1)RT-qPCR was used to detect the expression of circFoxo3.In early pregnant mice model,circFoxo3 was significantly lower at the D5,D6 and D7 implantation sites.In artificially induced decidualization mice model and stromal cells in vitro decidualization model,the expression of circFoxo3 was significantly reduced after decidualization induced.(2)Up-regulation of circFoxo3 inhibit stromal cells decidualizationUp-regulated circFoxo3 expression in stromal cells by transfection with circFoxo3 overexpression vector,and RT-qPCR results showed the expression of decidualization marker Hoxa10 was significantly reduced and F-actin staining showed cellular morphology had no obvious decidualization change after in vitro decidualization.(3)Prediction and verification target miRNA-mRNA of circFoxo3Bioinformatics analysis revealed circFoxo3 can bind miR-138-5p,and the binding of circFoxo3 and miR-138-5p has been verified in published paper using Dual luciferase reporter analysis.Bioinformatics analysis revealed miR-138-5p can bind Klf11 3'UTR region,and the binding of miR-138-5p and Klf11 was verified by Dual luciferase reporter analysis.(4)Up-regulation of circFoxo3 by transfection with circFoxo3 overexpression vector,RT-qPCR and western results showed the expression of miR-138-5p was significantly reduced and the expression of Klf11 protein was significantly increased after in vitro decidualization.(5)Up-regulation of miR-138-5p by miR-138-5p mimic transfection,western blot results showed the expression of Klf11 protein was significantly reduced after in vitro decidualization.(6)Up-regulation of Klf11 inhibit stromal cells decidualizationWestern blot were used to detect the expression of Klf11 protein.In stromal cells in vitro decidualization model,the expression of Klf11 was significantly reduced after decidualization induced.Up-regulated Klf11 expression in stromal cells by transfection with Klf11 overexpression vector,RT-qPCR results showed the expression of decidualization marker Hoxa10 was significantly reduced and F-actin staining showed cellular morphology had no obvious decidualization change after in vitro decidualization.Conclusion:The study revealed distinguishable expression pattern of circRNA between implantation sites and inter implantation sites at D5 pregnant mice endometrium.circFoxo3 was essential for endometrial stromal cells decidualization,and the potential mechanism in decidualization need to be further explored.