沉默miR-6771-3p通过靶向AXIN2/Wnt信号通路调控异位子宫内膜间质细胞增殖和迁移实验研究

Silencing miR-6771-3p regulates the proliferation and migration of ectopic endometrial stromal cells by targeting AXIN2/Wnt signaling pathway

目的:探讨微小RNA(miRNA)miR-6771-3p在子宫内膜异位组织中的表达及其通过靶向调控AXIN2对异位子宫内膜间质细胞增殖和迁移的影响。方法:收集因子宫内膜异位症行手术治疗的患者子宫在位内膜组织和异位内膜组织34例,采用实时聚合酶链式反应(RT-qPCR)检测组织中miR-6771-3p的表达水平。将异位子宫内膜间质细胞分为沉默组(转染靶向沉默miR-6771-3p的短发夹RNA载体)和空载体组(转染对照载体)。噻唑蓝(MTT)法分析转染后各组异位子宫内膜间质细胞的增殖能力,细胞划痕愈合实验分析转染后各组异位子宫内膜间质细胞的迁移能力,生物信息学技术和双荧光素酶报告基因实验验证miR-6771-3p和轴抑制蛋白2(AXIN2)的靶向关系,RT-qPCR检测转染后各组细胞AXIN2 mRNA表达水平。Western blot检测转染后各组细胞AXIN2蛋白和Wnt信号通路蛋白的表达水平。结果:子宫异位内膜组织中miR-6771-3p表达显著高于子宫在位内膜组织(P<0.01)。与空载体组异位子宫内膜间质细胞比较,沉默组异位子宫内膜间质细胞增殖能力显著降低(P<0.01),细胞划痕愈合率显著降低(P<0.01)。双荧光素酶报告基因实验证实miR-6771-3p与AXIN2存在靶向关系(P<0.01),miR-6771-3p能够负调控AXIN2 mRNA表达(P<0.01)。与空载体组相比,沉默组细胞中AXIN2蛋白表达显著升高,Wnt信号通路蛋白表达显著降低(均P<0.01)。结论:在子宫异位内膜组织中miR-6771-3p呈高表达,miR-6771-3p通过靶向AXIN2基因影响Wnt信号通路转导,进而调控异位子宫内膜间质细胞的增殖和迁移。

Objective:To investigate the expression of microRNA(miR)-6771-3p in ectopic endometrial tissue and its effect on the proliferation and migration of ectopic endometrial stromal cells by targeting and regulating AXIN2.Methods:34 cases of eutopic endometrial tissue and ectopic endometrial tissue of patients undergoing surgical treatment for endometriosis were collected,and the expression level of miR-6771-3p in tissues was detected by Real-time polymerase chain reaction(RT-qPCR).The ectopic endometrial stromal cells were divided into silence group(transfected with short hairpin RNA vector for silencing miR-6771-3p)and empty vector group(transfected with control vector).MTT assay was used to analyze the proliferation ability of endometrial stromal cells in each group after transfection.Cell scratch healing assay was used to analyze the migration ability of ectopic endometrial stromal cells in each group after transfection.Bioinformatics technology and dual luciferase reporter gene experiments verified the targeting relationship between miR-6771-3p and AXIN2.The expression levels of AXIN2 mRNA in each group of cells after transfection were detected by RT-qPCR.Western blot was used to detect the expression of AXIN2 protein and Wnt signaling pathway protein in each group of cells after transfection.Results:The expression of miR-6771-3p in ectopic endometrium tissue was significantly higher than that in eutopic endometrium tissue(P<0.01).Compared with the ectopic endometrial stromal cells in the empty vector group,the proliferation ability of the ectopic endometrial stromal cells in the silence group was significantly reduced(P<0.01),and the cell scratch healing rate was significantly reduced(P<0.01).Dual luciferase reporter gene experiments confirmed that there was a targeting relationship between miR-6771-3p and AXIN2(P<0.01),and miR-6771-3p could negatively regulate the expression of AXIN2 mRNA(P<0.01).Compared with the empty vector group,the expression of AXIN2 protein in the cells of the silencing group was significantly increased,and the expression of Wnt signaling pathway protein was significantly decreased(all P<0.01).Conclusion:miR-6771-3p is highly expressed in endometriosis tissue,and miR-6771-3p affects the transduction of Wnt signaling pathway by targeting AXIN2 gene,and then regulates the proliferation and migration of ectopic endometrial stromal cells.