Effect Of ADAR1 On The Biological Function Of Eutopic Endometrial Cells In Endometriosis

Endometriosis(EMs)is an estrogen-dependent gynecological disease in which the bioactive endometrial tissue appears in other parts of the uterine cavity.The main metastatic site is the pelvic peritoneum,ovary and vaginal rectum depression.The clinical manifestations of the disease are various,with dysmenorrhea,infertility,and abnormal menstrual flow as the main symptoms.In recent years,the incidence of endometriosis has been on the rise,and it has become one of the most common gynecological diseases among women of childbearing age.Endometriosis is a benign lesion in histopathology,but has the biological characteristics of malignant tumors,such as infiltration,metastasis and recurrence,and a few patients may develop malignant transformation.Studies have shown that endometriotic ectopic foci and some adjacent gynecological tumors have common genetic alterations,and gene spectrums for malignant transformation.RNA editing is an important post-transcriptional modification that can cause genetic information changes and protein diversity.In humans,the most common type of RNA editing is A-to-I editing,which is catalyzed by adenosine deaminase family(ADARs)acting on double-stranded RNA(dsRNA).The ADARs family consists of three members: ADAR1,ADAR2 and ADAR3.ADAR1 and ADAR2 are ubiquitously expressed in tissues with RNA editing activity,while ADAR3 is expressed only in brain tissue without editing activity.At present,ADAR1 is an adenosine deaminase with the most specific research and biological function.ADAR1-mediated misregulation of A-to-I RNA editing is associated with tumorigenesis by inactivating tumor suppressor or activating genes that promote tumor progression.In cell and mouse model studies,ADAR1 overexpression increased tumor-associated characteristics such as cell proliferation,migration,and invasion,and down-regulation of ADAR1 reduced these characteristics,suggesting that ADAR1 promotes cell proliferation,migration,and invasion.Endometriosis has some biological characteristics similar to tumors,so we suspect that ADAR1 will also have an impact on the development of endometriosis.So far,studies on the relationship between ADAR1 and endometriosis have not been reported.ObjectiveStudy the effect of ADAR1 on the biological function of eutopic endometrial cells in endometriosis to explore the role of ADAR1 in the development of endometriosis.Material and methods 1.Study populationThe endometrial tissues of patients undergoing hysterectomy in the Third Affiliated Hospital of Zhengzhou University from November 2017 to April 2018,including 6 cases normal endometrial tissues(NE)and 6 cases eutopic endometrial tissues(EE)of endometriosis,were selected.The above specimens were obtained during the endometrial proliferative phase.Inclusion criteria: Patients were not treated with hormones for 3 months before surgery,aged between 35 and 49 years,and regular menstruation.Exclude other endometrial diseases,such as endometritis,endometrial cancer,etc.Groups: endometriosis eutopic endometrial cells were divided into ADAR1-shRNA experimental group,shRNA negative control group(shRNA-NC group),blank control group(EE group);normal endometrial cells were divided into ADAR1 overexpression group(ADAR1-OE group),the overexpression negative control group(OE-NC group),and the blank control group(NE group).2.Methods(1)The collected specimens were immediately placed in sterile physiological salinesupplemented with a double antibody for cell culture.(2)ADAR1 overexpression plasmid and ADAR1 silencing plasmid were constructedand transfected into normal endometrial cells and eutopic endometrial cells ofendometriosis.(3)The expression of ADAR1 mRNA and ADAR1 protein in endometrial cells ofeach group was detected by RT-qPCR and Western blot.(4)The cell viability of endometrial cells in each group was detected by CCK-8method,and the proliferation ability of endometrial cells was detected by EdUmethod.(5)The invasion and migration ability of endometrial cells in each group weredetected by Transwell method.The activity of metalloproteinase-9(MMP-9)inendometrial cells was identified by gelatin zymography.(6)The apoptosis ability of endometrial cells was detected by flow cytometry.3.Statistical analysisThe experimental data were expressed as mean ± standard deviation,and statistical analysis was performed using SPSS 21.0 software.For the measurement data that accorded with normality and homogeneity of variance,the t test was used for comparison between the two groups,and one-way ANOVA was used for comparison among groups.The test level is ?=0.05.P < 0.05 was considered as statistical difference.Results 1.ADAR1 overexpressed and silencing cell line constructionRT-qPCR and Western blot showed that the mRNA and protein expression levels of ADAR1 in ADAR1-OE group were significantly higher than those in OE-NC group(P<0.05).The expression of ADAR1 mRNA and protein in ADAR1-shRNA group was significantly lower than that in shRNA-NC group(P<0.05).2.Effect of ADAR1 on endometrial cell viability and proliferationThe results of CCK-8 showed that the OD450 nm value of ADAR1-OE group was significantly higher than that of OE-NC group(P<0.01).The OD450 nm values of ADAR1-shRNA1 and ADAR1-shRNA2 were significantly lower than those of shRNA-NC(P<0.001).The Edu results showed that the average cell proliferation of ADAR1-OE group was significantly higher than that of OE-NC group(P<0.01).The average cell proliferation of the ADAR1-shRNA1 and ADAR1-shRNA2 groups was significantly lower than that of the shRNA-NC group(P < 0.001).3.Effect of ADAR1 on endometrial cell invasion and migrationThe results of Transwell showed that the number of cells passing through the basement membrane of the ADAR1-OE group was significantly higher than that of the OE-NC group(P<0.01);The number of cells in the ADAR1-shRNA group across the artificial basement membrane was significantly lower than in the shRNA-NC group(P < 0.001).Gelatin zymography assay results showed that the expression of MMP-9 in ADAR1-OE group was higher than that in OE-NC group(P<0.05).MMP-9 expression was lower in the ADAR1-shRNA1 and ADAR1-shRNA2 groups than in the shRNA-NC group(P<0.05).4.Effect of ADAR1 on endometrial cell apoptosisFlow cytometry results showed that there was no significant difference between the ADAR1-OE group and the OE-NC group(P>0.05),but the apoptotic rate of ADAR1-shRNA was significantly higher than that of shRNA-NC group(P<0.05).Conclusions(1)The expression of ADAR1 in eutopic endometrial cells of endometriosis was significantly higher than that of normal endometrial cells;(2)ADAR1 can increase the viability and proliferation of endometrial cells.(3)ADAR1 enhances the invasion and migration of endometrial cells,and this increase in capacity may be associated with increased MMP-9 activity.(4)The effect of ADAR1 on the apoptotic ability of endometrial cells showed that there was no significant change in the apoptosis of endometrial cells after overexpression of ADAR1 in normal endometrial cells.After silencing ADAR1 in endometrial cells of endometriosis,cell apoptosis is significantly increased.