The Expression And Function Of ADAR1 In Eutopic And Ectopic Endometrium Of Women With Endometriosis

Endometriosis is a common gynecological disease,characterized by endometrial-like tissue growing outside uterus,for example ovary,fallopian tube,pelvic cavity and abdominal cavity,with ovary the most prevalent place.The clinical manifestation varied in severity and symptoms,including irregular menstruation,chronic abdominal pain irrelevant of menstrual cycle,dyspareunia,hypermenorrhea,hypogastralgia,low fertility or infertility.Adenomyosis is a gynecological disease caused by endometrial glands and stroma growing into myometrium,characterized by hypermenorrhea,menostaxis,and secondary dysmenorrhea.Adenomysis is widely thought to be a type of endometriosis.They share common pathogenesis in general.Though endometriosis and adenomysis are non-tumorous diseases,they share some common characteristics with malignant tumor,for example,abnormal proliferation,infiltration or even invasion.Recent studies have shown that cell immunology and humoral immune disorders have something to do with endometriosis and adenomyosis development.There are no reports about ADAR1 in endometriosis,nor in adenomyosis.ADAR1 is a RNA editing enzyme.RNA editing means to modify and process RNA after transcription from DNA,which may differ the expression of protein level from DNA level,thus changing genetic information.Research has shown that ADAR1 plays a very important role in cell proliferation,diffraction and tumor progression.In addition,ADAR1 has a close relation with inflammation and immunology.In this research,we use Real-time PCR,Western blotting and immunohistochemistry to detect the expression of ADAR1 endometriosis and adenomyosis.To explore the possible pathogenesis of the two diseases and may provide potential novel clinical treatments.ObjectiveDetect ADAR1 expression in both endometriosis patients and adenomyosis patients.Explore the relationship between ADAR1 with endometriosis and adenomyosis.Materials and MethodsThe study was approved by the local Ethics Committee of Third Affiliated Hospital of Zhengzhou University.Written informed consent was obtained from each patient.1 Study PatientsRecruited patients had regular menstrual cycles(24-35days),and in the proliferative phase of their menstrual cycle.The Endometriosis study group included patients(n=31)with endometriosis who underwent laparoscopic excision of ovarian endometrioma,and were classified as having stage III or IV endometriosis according to the American Society for Reproductive Medicine classification of endometriosis(The American Fertility Society,1985).The adenomyosis study group included patients(n=30)undergoing hysterectomy.Control group(n=30)are patients with unexplained infertility processing diagnostic curettage or undergoing hysterectomy because of other disease.None of the women had taken any hormonal therapy and GnRH anologs in the past six months.2 Sample collectionEach sample,some put in liquid nitrogen immediately and put in-80? freezers for RT-PCR and Western Blotting use,the others put in 10% formaldehyde solution and embedded in paraffin for histological examination.3 Method1.Using Real-time PCR(qRT-PCR)to detect ADAR1 mRNA in each group.2.Using Western blotting to semiquantitative ADAR1 protein in each group.3.Using lab imaging analysis system to semiquantitative ADAR1 expression in immunohistochemical staining slides.4 Statistic analysisStatistical analysis was performed using SPSS 21.0 software(IBM,Chicago,Illinois).The quantitative data are shown as mean+standard error of the mean(SEM),and the results were analyzed using one-way analysis of variance method or rank sum test for the unequal variance.A P value <.05 was considered statistically significant.Result 1 Comparing age of each group,there is no significant statistic difference between groups.2 Expression of ADAR1 mRNA in endometriosis and adenomyosisReal-time RCR results show that ADAR1 P150 mRNA in EC(2.33±0.28)and EU group(2.18±0.36)is much higher than C group(1.68±0.43)(P<0.05).But there is no significant difference of P150 mRNA expression between EU group and EC group.Also,there is no significant difference of P110 mRNA expression between EU group(1.01±0.21),EC group(1.01±0.15)and C group(0.99±0.11)(P>0.05).Similar to EMs results,RT-PCR results show that ADAR1 P150 mRNA in AM1 group(2.09±0.26)and AM2 group(2.09±0.25)is significantly higher than control group(1.68±0.43)(P<0.05),while there is no significant difference of ADAR1 P110 2 Sample collection mRNA expression between AM1 group(0.97±0.21),AM2 group(0.87±0.17)and C group(0.99±0.11).3 ADAR1 protein expression in endometriosis and adenomyosisWestern blotting results show that ADAR1 protein expression is much higher in EU group(0.39±0.05)and EC group(0.40±0.07)than in C group(0.104±0.04)(P<0.05),but there is no significant difference between EU group and EC group(P=0.517).The expression of ADAR1 protein in AM1(0.34±0.06)and AM2 group(0.35±0.06)is much higher than control group(0.104±0.04)(P<0.05),while there is no significant difference of ADAR1 protein expression between AM1 and AM2(P>0.05).3 Immunohistochemistry resultsImmunohistochemistry results showing that there is brown positive staining in cytoplasm in endometrium of all groups,the ADAR1 expression in EU(87.29±11.87),EC(96.65±13.31)and C group(26.08±8.49)(p<0.05),while in AM1,AM2 and C group(68.77±11.20),(67.48±11.44),(26.08±8.49).ConclusionsADAR1 may be a critical factor in the pathogenesis of endoendometriosis and adenomyosis,which is a new clue for searching a new method of diagnosis and treatment of EMs and AM.