The Mechanism Of The Regulation Of Endometrial Receptivity By Nucleolar Small Molecule RNA SnoRA75

ObjectivesEndometrial receptivity is a complex process,which provides the embryo with the opportunity of attachment,invasion and development,and finally forms a new individual and species continuity.Endometrial receptivity refers to "endometrial maturation",during which the trophoblastic ectoderm of the blastocyst can attach to the endometrial epithelial cells,and then enter the endometrial stroma and vascular system".Endometrial receptivity is an important barrier of assisted reproductive technology.Scientific understanding of endometrial receptivity is the basis of understanding human reproduction,but up to now,no biochemical index of endometrial receptivity has been proved to be clinically useful.The mechanism of uterine function,especially the regulation of endometrial receptivity,has been widely studied in the past two decades.In recent years,a large number of non-coding RNAs(ncRNAs)with various structures and functions are related to a variety of human diseases,including cancer.Small nucleolar RNA(SnoRNA)is an important player in the regulation of gene expression in human cells.The regulatory functions of box C/D and box H/ACA SnoRNA are post transcriptional modification of ribosomal RNA(rRNA),2-O-methylation and pseudouridine,respectively.A series of independent studies have shown that SnoRNA and other non-coding RNAs can be used as sources of various short regulatory RNAs.Some SnoRNAs and their fragments can also be involved in the regulation of mRNA alternative splicing and post transcriptional modification.Changes in the expression of SnoRNA in human cells can affect many important cellular processes.The level of SnoRNA in human cells,serum and plasma is a promising target for the diagnosis and treatment of human pathology.Small nucleolar RNAs(SnoRNAs)are the most abundant class of ncRNAs encoded by introns.SnoRNA plays an important role in human physiological and pathological processes.This study will focus on the regulatory role of SnoRNA in endometrial receptivity.Materials and methods1.High throughput sequencing was used to sequence and analyze endometrial tissues of different periods.RNA was extracted from samples,and the expression of SnoRNA in endometrial tissues of different periods was verified by real-time fluorescent quantitative PCR.The localization of SnoRA75 in cells was detected by FISH experiment.We detected the effect of reproductive hormones on SnoRA75 expression in EECs.2.We constructed lentiviruses that overexpressed or knocked down SnoRA75 that were transfected into endometrial cells,and detected the expression of SnoRA75 in endometrial cells by FISH and real-time fluorescent quantitative PCR.CCK-8,flow cytometry,cell scratch and Transwell were used to detect the effect of SnoRA75 on the proliferation,apoptosis,migration and invasion of endometrial cells by real-time fluorescence quantitative PCR and Western blot.The influence of SnoRA75 on the expression of receptivity related factors was detected by real-time fluorescence quantitative PCR and Westernblot.We constructed an in vitro adhesion model to evaluate the effect of SnoRA75 on the adhesion rate of trophoblasts in vitro.To further study the effect of SnoRA75 on embryo implantation,we used lentivirus to regulate the expression of SNORA75 in mice.3.MiRNA combined with SnoRA75 was analyzed by bioinformatics software.RNA pull Down and luciferase reporter genes were used to detect the binding of SnoRA75 and miR-146a-3p.MiR-146a-3p overexpression lentivirus was constructed in endometrial cells,and miR-146a-3p knockdown lentivirus was constructed to detect the expression of miR-146a-3p in endometrial cells.CCK-8,flow cytometry,cell scratch and Transwell were used to detect the proliferation of miR-146a-3p in endometrial cells.The effects of miR-146a-3p on the expression of tolerance related factors were detected by real-time fluorescence quantitative PCR and Western blot.We constructed an in vitro adhesion model to investigate the effect of miR-146a-3p on the adhesion rate of trophoblasts in vitro.To further study the effect of miR-146a-3p on embryo implantation,we used miR-146a-3p miRNA mimics and inhibitors to regulate the expression of miR-146a-3p in mice.4.The target gene of miR-146a-3p was predicted by targetscan.The luciferase reporter gene was constructed to verify the binding of miR-146a-3p with the target gene.Western blot was used to detect the binding of miR-146a-3p with the target gene which was regulated by miR-146a-3p.The influence of miR-146a-3p overexpression and inhibition on the expression of ZNF23 were detected by real-time fluorescence quantitative PCR and westernblot.The influence of ZNF23 on the expression of receptivity related factors was detected by real-time fluorescence quantitative PCR and Westernblot.Westernblot was used to detected the influence of SnoRA75 overexpression and inhibition on the expression of ZNF23.We detected the interaction and influence of SnoRA75,miR-146a-3p and ZNF23 by Westernblot.5.Exosomes were isolated by ultracentrifugation.The interaction of SnoRA75 and receptivity related factors was detected by real-time fluorescent quantitative PCR.Results1.There were 37 different expression of SnoRNA,13 of which were significantly higher in secretory metaphase compared with proliferative phase,and 24 of which were significantly lower in secretory metaphase compared with proliferative phase(P<0.05).Real-time fluorescent quantitative PCR showed that the expression of SnoRA75 was significantly higher in secretory metaphase compared with proliferative phase(P<0.01).E2,P4 and HCG can induce the high expression of SnoRA75 in endometrial cells(P<0.01).FISH detection showed that SnoRA75 was mainly located in the cytoplasm;2.The overexpression of slow virus SnoRA75 can significantly promote the proliferation,migration and invasion of endometrial cells(P<0.01).The knockdown of slow virus SnoRA75 can significantly inhibit the proliferation,migration and invasion of endometrial cells(P<0.01).The overexpression of slow virus SnoRA75 has no effect on the apoptosis of endometrial cells and the knockdown of slow virus SnoRA75 could significantly promote the apoptosis of endometrial cells(P<0.01).The overexpression of slow virus SnoRA75 significantly promoted the expression of LIF,integrin3,claudin4 and DKK1,and the knockdown of slow virus SnoRA75 significantly inhibited the expression of LIF,integrin3,claudin4 and DKK1 in endometrial cells by real-time fluorescent quantitative PCR and Western blot(P<0.01).The adhesion rate of the pLKO.1-SnoRA75 group was significantly decreased(P<0.01)and SnoRA75 overexpression significantly improved the adhesion rate(P<0.05).SnoRA75 overexpression effectively promoted embryo implantation,while the inhibition of SnoRA75 expression significantly inhibited embryo implantation(P<0.05).3.The results of bioinformatics software analysis and RNA pull down showed that miR-146a-3p could be targeted by SnoRA75.The overexpression of miR-146a-3p by lentivirus could significantly inhibit the proliferation,migration and invasion of endometrial cells(P<0.05).The inhibition of miR-146a-3p can significantly promote the proliferation,migration and invasion of endometrium(P<0.05).The inhibition of miR-146a-3p had no effect on the apoptosis of endometrial cells,and the overexpression of miR-146a-3p could significantly promote the apoptosis of endometrial cells(P<0.05).MiR-146a-3p overexpression inhibited the expression of LIF,integrin3,claudin4 and DKK1,and inhibition of miR-146a-3p expression significantly promoted the expression of LIF,integrin3,claudin4 and DKK1 in endometrial cells by real-time fluorescent quantitative PCR and Western blot(P<0.01).The adhesion rate of the miR-146a-3p mimic group was significantly lower than that of the miRNA mimic NC group(P<0.05),and inhibition of miR-146a-3p significantly improved the adhesion rate(P<0.01).MiR-146a-3p overexpression inhibited the implantation of embryos,and inhibition of miR-146a-3p expression significantly promoted embryo implantation(P<0.05).4.The results of bioinformatics software analysis showed that miR-146a-3p could be targeted by ZNF23.The results of luciferase reporter gene and Western blot showed that miR-146a-3p could target and regulate the function of ZNF23(P<0.05).MiR-146a-3p overexpression significantly inhibited the expression of ZNF23 protein,and miR-146a-3p inhibition significantly promoted ZNF23 expression by real-time fluorescent quantitative PCR and Western blot(P<0.05).Western blot analysis showed that SnoRA75 overexpression significantly promoted ZNF23 expression,and SnoRA75 inhibition significantly inhibited ZNF23 expression(P<0.05).Western blot analysis showed that when the function of miR-146a-3p was inhibited,the SnoRA75-mediated promotion of ZNF23 expression was lost,indicating that this effect was miR-146a-3p dependent.ZNF23 overexpression significantly promoted the expression of LIF,integrin3,claudin4 and DKK1,and silencing ZNF23 significantly inhibited the expression of LIF,integrin3,claudin4 and DKK1 in endometrial cells by real-time fluorescent quantitative PCR and Western blot(P<0.05).5.LIF stimulates endometrium to release exosomes rich in SnoRA75.Endometrial exosomes can significantly promote the expression of receptive factors in endometrial cells(P<0.05).Conclusion1.The expression of SnoRA75 in different stages of endometrium has time sequence.SnoRA75 was highly expressed in the secretory phase of endometrium.E2、P4、HCG can induce SnoRA75 expression in endometrial cells.2.The overexpression of SnoRA75 could significantly promote the proliferation,migration and invasion of endometrial cells.SnoRA75 can significantly promote endometrial receptivity.3.The overexpression of miR-146a-3p could significantly inhibit the proliferation,migration and invasion of endometrial cells.MiR-146a-3p suppresses endometrial receptivity.4.Through the SnoRA75/miR-146a-3p/ZNF23 signaling pathway,SnoRA75 regulates the function of endometrial cells and endometrial receptivity.5.Endometrial exosomes can significantly promote the expression of receptive factors in endometrial cells.