CircPTPN12/miR-21-5p/ΔNp63α Axis Dysregulation Contributing To Endometrial Fibrosis

Background: Intrauterine adhesions(IUA)is a disease caused by injury to the basal layer of the endometrium resulting in hypomenorrhea even amenorrhea,infertility or recurrent spontaneous abortion and the leading cause of uterine infertility.Current treatments play a limited role in ameliorating endometrial fibrosis for patients with severe IUA and many patients occur recurrent intrauterine adhesions postprocedure.IUA is characterized by endometrium fibrosis but the detailed mechanism remains unclear.Micro RNAs(mi RNAs)play essential roles in various fibrotic diseases,however their role in endometrial fibrosis is just beginning to unravel.Among regulators for mi RNAs,circular RNAs(circ RNAs)rank in the front.Circ RNAs have been shown to participate in a variety of physiological and pathological processes,but the regulatory role in IUA has not been reported so far.Our previous research found that the abnormal upregulation of ΔNp63α in endometrium of IUA patients promotes epithelial-mesenchymal transition(EMT).In addition,mi RNAs are important regulators of ΔNp63α.Therefore,we screened out mi R-21-5p and circ PTPN12 by highthroughput sequencing in IUA and explored whether dysregulation of circ PTPN12/mi R-21-5p/ ΔNp63α axis contributes the pathogenesis of IUA.Objective: To confirm the existence and role of the circ PTPN12/ mi R-21-5p/ ΔNp63αregulatory axis in the pathogenesis of IUA and provide an effective therapeutic target for IUA.Methods: 1.Differential expression of mi RNAs in endometrium of IUA patients and normal controls were detected by high-throughput sequencing.The expression of mi R-21-5p was verified by real-time quantitative PCR(q RT-PCR)and RNAscope technology.The targeting relationship between mi R-21-5p and ΔNp63α was predicted by the micro RNA database and verified by dual luciferase reporter and Western blotting.We also detected the expression of EMT and fibrosis indicators in endometrial epithelium cells(EECs)with overexpression or knockout of mi R-21-5p or ΔNp63α.2.The expression characteristics of circ RNAs that bind to mi R-21-5p in the endometrium of IUA patients were predicted by the mi Randa database and screened by high-throughput sequencing.The expression of circ PTPN12 was verified by q RT-PCR.The binding relationship between circ PTPN12 and mi R-21-5p was verified by RNA pull-down,dual luciferase reporter,fluorescence in situ hybridization and RNA immunoprecipitation experiments.The effect of circ PTPN12/ mi R-21-5p/ ΔNp63α in EEC-EMT were also detected.3.In vivo experiments further explored the changes of EMT and fibrosis indexes caused by overexpression of circ PTPN12 or mi R-21-5p in the mechanically injured endometrium of IUA mice model.Results: 1.The expression of mi R-21-5p was the most abundant in normal endometrium while significantly reduced in endometrium of patients with severe IUA.Mi R-21-5p targeted to the 3’ UTR region of ΔNp63α and exerted anti-EEC-EMT role by suppressing the expression of ΔNp63α.2.The expression of circ PTPN12 was significantly increased in the endometrium of patients with severe IUA.Circ PTPN12 was derived from 5-8 exons of PTPN12 gene and had mi R-21-5p binding site.Circ PTPN12/ mi R-21-5p/ ΔNp63αregulatory axis was present and circ PTPN12 acted as a functional sponge of mi R-21-5p to regulate ΔNp63α expression and EEC-EMT.3.Circ PTPN12 aggravated the EMT and endometrial fibrosis induced by mechanically injuring in vivo.Mi R-21-5p can alleviate circ PTPN12-mechanicalinduced EEC-EMT in IUA mice model.Conclusions: Circ PTPN12,which is highly expressed in the endometrium of patients with severe IUA,serves as a competing endogenous RNAs(ce RNA)by competitively bounding to mi R-21-5p,then abolishes suppressive effect of mi R-21-5p on its target gene ΔNp63α,resulting in the EEC-EMT and endometrium fibrosis.The mechanism of circ PTPN12/ mi R-21-5p/ ΔNp63α axis dysregulation in IUA provides a new intervention target for the treatment of IUA.