| Endometriosis(EMs)is a benign disease,but it has the characteristics similar to malignant tumor,such as implantation,distant invasion and metastasis.Endometriosis refers to the presence of endometrial tissue(glands and stroma)outside the uterine body,which has a serious impact on the physical and mental health of women,and has become a worldwide troubling disease.In view of endometriosis,scholars from domestic and foreign countries have a large number of studies,putting forward a variety of hypotheses,but its pathogenesis is complex,the exact mechanism has not been fully elucidated.Some studies have found that the biological behavior of eutopic endometrium is similar to that of ectopic endometrium."Eutopic endometrium determinism" suggests the biological properties of EMs eutopic endometrial stromal cells,such as greater ectopic implantation,invasion and more likely to developing lesions at a distance.miRNAs may be involved in the pathogenesis of endometriosis.As posttranscriptional regulators,miRNAs,approximately 18-25nt in length,bind to the 3’non-coding regions(3’UTR)of target mRNAs in an incomplete base complementary pair to control posttranscriptional gene expression.Previous studies have shown that miR-363 can participate in the regulation of multiple life activities,such as proliferation,apoptosis and invasion,and its expression is low in endometriosis.Therefore,it is speculated that miR-363 also plays a certain role in the occurrence and development of endometriosis.In this study,the expression level of miR-363 in endometrial stromal cells was changed by cell transfection to explore whether it has an impact on the biological behavior of endometrial stromal cells in endometriosis.ObjectiveTo explore the effect of miR-363 on the biological behavior of EMs endometrial stromal cells,the EMs eutopic endometrial stromal cells and normal eutopic endometrial stromal cells were isolated and cultured.The expression level of miR-363 was changed by cell transfection,which is used to compare the biological activity bewteen EMs eutopic endometrium and normal endometrium,and study the effect on the biological behavior of endometrium stromal cells when the expression of miR-363 was changed.Materials and Methods1 Research object and experimental groupsEMs eutopic endometrium were collected from 32 patients who underwent surgery for endometriosis in the Third Affiliated Hospital of Zhengzhou University from January 2019 to December 2019.The ASRM modified endometriosis staging method(1997)was used.2 patients in stage Ⅰ-Ⅱ and 30 in stage Ⅲ-Ⅳ.All 32 patients had ovarian endometriosis,21 patients had peritoneal endometriosis only,1 patient had deep infiltrating endometriosis,and 8 patients had both.The normal endometrium was collected from 32 patients who underwent hysterectomy due to cervical intraepithelial neoplasia in our hospital during the same period.All patients had regular menstruation(the menstrual cycle is 21-35 days and the menstrual period is 3-7 days),and was confirmed by pathology after operation.No GnRH analogues or hormone drugs were received in 3 months before surgery.No other internal and surgical diseases,endocrine diseases or tumors.No endometrial disease and other gynecological diseases.Partial endometrium of each group was obtained by aseptic curettage during operation,washed with normal saline,and transported to the laboratory within 1 hour.This study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University,and all patients signed informed consent.This study was divided into 6 groups.EMs eutopic endometrial stromal cells were divided into three groups,including m-363 group(transfection with miR-363 mimics),NC group(transfection with mimics negative control),and the ESC group(non-transfected negative control).The Lipofectamine 2000 transfection reagent was used for transfection.The normal endometrial stromal cells in the patients without EMs were divided into three groups,including in-363 group(transfection with miR-363 inhibitor),iNC group(transfection with inhibitor negative control),and NSC group(non-transfected negative control).The Lipofectamine 2000 transfection reagent was used for transfection.2 experimental methodThe EMs eutopic endometrial stromal cells and the normal endometrial stromal cells of the patients without EMs were isolated and cultured.The expression of miR-363 in endometrial stromal cells of each group was interfered by cell transfection technique,and the effect of miR-363 on the biological phenotype of eupotic endometrial stromal cells was studied by CCK-8,Flow cytometry,Transwell invasion.In addition,ICAM-1 was detected by ELISA to investigate the effect of miR-363 on the adhesion of endometrial stromal cells.3 Statistical analysisThe data was analyzed by GraphPad Prism8 software.All the result were expressed as mean ± standard deviation.Comparision between the two groups used the independent samples t-test.One-way ANOVA was used for data from three groups.Two-way ANOVA was used to compare the three groups at different time points.With α=0.05 for the test standards.Results1.Endometrial stromal cells were successfully isolated and cultured.2.In the EMs eutopic endometrial stromal cells,miR-363 was highly expressed in the m-363 group by cell transfection,and the difference was statistically significant compared with the NC group(P<0.001).In the normal endometrial stromal cells of the patients without EMs,the expression of miR-363 in the in-363 group was reduced by cell transfection,and the difference was statistically significant compared with that in the iNC group(P<0.001).3.The results of CCK-8 experiment showed that the relative proliferation activity in the non-transfected ESC group was higher than that of the non-transfected NSC group at 12h,24h,36h and 48h after culture,and the difference was statistically significant(P<0.05).In the EMs eutopic endometrial stromal cells,after transfection with miR-363 mimics,the relative proliferation activity in the m-363 group was significantly lower than that in the NC group and the non-transfected ESC group at 12h,24h,36h and 48h after culture,and the differences were statistically significant(P<0.001).In the normal endometrial stromal cells of the patients without EMs,after transfection with miR-363 inhibitor,the relative proliferation activity in the in-363 group was significantly higher than that in the iNC group and the non-transfected NSC group at 12h,24h,36h and 48h after culture with miR-363 inhibitor,and the differences were statistically significant(P<0.05).4.Flow cytometry showed that apoptosis rate in the non-transfected ESC group was significantly lower than that in the untransfected NSC group,and the difference was statistically significant(P<0.001).In the EMs eutopic endometrial stromal cells,after transfection with miR-363 mimics,the apoptosis rate in the m-363 group was significantly higher than that in the NC group,and the differences were statistically significant(P<0.01).In the normal endometrial stromal cells of the patients without EMs,after transfection with miR-363 inhibitor,the apoptosis rate in the in-363 group was significantly lower than that in the iNC group and the non-transfected NSC group,and the differences were statistically significant(P<0.001).5.The results of transwell experiments showed that the cell counts passing through the chamber in the non-transfected ESC group(585.50±24.58)was higher than that in the non-transfected NSC group(446.67±27.66),and the difference was statistically significant(P<0.001).In the EMs eutopic endometrial stromal cells,after transfection with miR-363 mimics,the cell counts passing through the chamber in the m-363 group(257.50±29.91)was significantly lower than that in the NC group(554.67±31.12)and the non-transfected ESC group,and the differences were statistically significant(P<0.001).In the normal endometrial stromal cells of the patients without EMs,after transfection with miR-363 inhibitor,the cell counts passing through the chamber in the in-363 group(535.50±24.69)was significantly higher than that in the iNC group(415.17±21.86)and the non-transfected NSC group,and the differences were statistically significant(P<0.001).6.ELISA experient results showed that the expression of ICAM-1 in non-transfected ESC group(1.81±0.21)was significantly higher than that in non-transfected NSC group(1.02±0.23),and the difference was statistically significant(P<0.001).In the EMs eutopic endometrial stromal cells,the expression of ICAM-1 in the m-363 group(1.04±0.18)was significantly lower than that in the NC group(1.78±0.21)and non-transfected ESC group,and the differences were statistically significant(P<0.001).In the normal endometrial stromal cells of the patients without EMs,the expression of ICAM-1 in the in-363 group(1.44±0.11)was significantly lower than that in the iNC group(1.03±0.13)and non-transfected NSC group,and the differences were statistically significant(P<0.01).ConclusionEMs ectopic endometrium has stronger proliferation activity and invasion activity,but the apoptosis ability and the expression of ICAM-1 were increased,namely to distant metastasis form lesions more easily.It will decrease the proliferation activity,enhance the apoptotic ability,weaken the migration ability,and reduce the expression of ICAM-1 when miR-363 is overexpressed in the EMs eutopic endometrial stromal cells.It will enhance the proliferation activity,decrase the apoptotic ability,enhance the migration ability,and increase the expression of ICAM-1 when the expression of miR-363 is inhibited.miR-363 can inhibit the proliferation and metastasis,promote apoptotic and reduce the expression of adhesion index ICAM-1 of eutopic endometrial stromal cells in endometriosis.miR-363 may play an inhibitory role in the pathogenesis of endometriosis,and may provide a target and theoretical basis for the treatment of endometriosis. |
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