Effects And Mechanisms Of MiR-363-3p On The Biological Properties Of Endometrial Stromal Cells

Background and objective:Endometriosis?EMs?is the most common benign disease of gynecology,which seriously affects women's health.The exact pathogenesis of endometriosis is still unknown,and there is still no effective treatment.MicroRNAs?miRNAs?are a group of noncoding RNAs?ncRNA?,which mainly control protein-coding genes by binding to the 3'UTR?Untranslated Region?of target mRNA.A large number of microRNAs have been found to play an important role in the occurrence of malignant tumor diseases and benign diseases including endometriosis.In previous research,we found that miR-363-3p is down-regulated and Methionine sulfoxide reductase B3?MsrB3?is up-regulated in ovarian endometriosis compared with eutopic endometrium by high-throughput sequencing techniques and MsrB3 is a predicted target of miR-363-3p.The purpose of this study was to investigate the effect of miR363-3p on the biological phenotype of endometrial stromal cells?EnSCs?and its mechanism.This study is divided into four parts.Section ?.Expression of miR-363-3p in eutopic endometrium?Eu?,ectopic endometrium?Ec?and normal endometrium?N?Objective:To investigate the expression of miR-363-3p in EMs.Methods:Eu?n=30?,Ec?n=3 7?tissues of EMs patients and N?n=30?tissues of normal control were collected.Using real-time quantitative reverse transcription PCR?qRT-PCR?detects the expression of miR-363-3p.Results:Compared with Eu,miR-363-3p expression was significantly lower in Ec.There was no significant difference in miR-363-3p expression between N and Eu.Conclusion:miR-363-3p expression was reduced in ectopic endometrium.Section ?.Isolation and culture of endometrial stromal cellObjective:To establish a suitable in vitro model of Ems.Methods:endometrial tissues of patients with EMs were collected.EnSCs were isolated and purified by enzyme digestion,filtration.Cell purity was identified by immunofluorescence.Results:the morphology and arrangement of EnSCs were in accordance with the literatures.The purity of EnSCs is above 90%.Conclusion:Qualified and cultured EnSCs can be used for furthe experiments in vitro.Section ?.The role of miR-363-3p on proliferation,apoptosis and invasion of endometrial stromal cellsObjective:To investigate the effect of miR-363-3p on proliferation,apoptosis and invasion ability of EnSCs in endometriosis.Methods:EnSCs were transfected with miRNA negative control?miR-NC?and miR-363-3p mimics by Lipofectamine 2000.The changes of miR-363-3p,Caspase3,ki-67,MMP-2 and MMP-9 after transfection were detected by qRT-PCR.Flow cytometry?FCM?and cell counting kit?CCK8?assay were used to detect the changes of apoptosis and proliferation capacity after overexpression of miR-363₃p in EnSCs.Results:Transfection of miR-363-3p mimics increased the expression of miR-363-3p in EnSCs,about 150 times that of the control group.After the transfection of miR-363-3p,the expression of caspase-3 increased,while the expression of ki-67 decreased.There were no significant changes in the expression of MMP-2 and MMP-9.miR-363-3p can promote EnSCs apoptosis and inhibit proliferation.Conclusion:miR-363-3p can reduce the vitality of EnSCs,thereby inhibiting the occurrence of endometriosis.Section ?.Verification of the potential target gene of miR-363-3p:methionine sulfoxide reductase B3?MsrB3?Objective:To investigate the relationship between miR-363-3p with MsrB3 and its possible mechanism involved in the pathogenesis of endometriosis.Methods:qRT-PCR and Western blot were used to detect the mRNA and protein expressions of MsrB3 in endometrial tissues of patients with or without endometriosis.Using luciferase reporter assay and western blot,we validate MsrB3 is a target gene of miR-363-3p from the aspects of binding ability and correlation respectively.The biological effects of MsrB3 on EnSCs were detected by CCK-8 and FCM assay after transfection of MsrB3 overexpressed plasmid.Results:Double luciferase reporter assay confirmed that miR-363-3p and MsrB3 have the ability to bind.Overexpression of miR-363-3p results in reduction expression of MsrB3 protein,but not MsrB3 mRNA.Overexpression of MsrB3 can reverse the decrease of cell proliferation and increase of apoptosis rate induced by miR-363-3p.Conclusion:MsrB3 is the target gene of miR-363-3p.miR-363-3p inhibits EnSCs proliferation and induces apoptosis which may be attributed to MsrB3.