miR-155在转化生长因子β2诱导的人视网膜色素上皮细胞上皮-间质转化中的促进作用  

Promotion effect of miR-155 on transforming growth factorβ2-induced epithelial-mesenchymal transition in human retinal pigment epithelial cells

作　　者：王艳婷 金学民[2] 李晓华[1] 赵朝霞[1] Wang Yanting;Jin Xuemin;Li Xiaohua;Zhao Zhaoxia(Department of Ophthalmology,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,Zhengzhou 450003,China;Department of Ophthalmology,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]河南省人民医院眼科,河南省立眼科医院,河南省眼科研究所,郑州450003 [2]郑州大学第一附属医院眼科,450052

出　　处：《中华实验眼科杂志》2021年第1期13-19,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：河南省自然科学基金项目(162300410296)。

摘　　要：目的探讨微小RNA-155(miR-155)在转化生长因子β2(TGF-β2)诱导的人视网膜色素上皮细胞上皮-间质转化过程中的作用及其机制。方法取视网膜色素上皮细胞ARPE-19细胞系分为对照组和TGF-β2组,分别用DMEM培养液和含10 ng/ml TGF-β2的DMEM培养液培养,另取ARPE-19细胞系分为miR-155抑制剂组和miR-155阴性对照组,分别用含miR-155抑制剂和miR-155阴性对照序列转染细胞,并在含10 ng/ml TGF-β2的DMEM培养液中培养,于培养后48 h采用逆转录PCR法检测细胞中miR-155表达,采用细胞划痕实验、Transwell小室实验分别检测细胞迁移、侵袭能力,采用Western blot法检测细胞中磷酸酶及张力蛋白同源物(PTEN)、磷酸肌醇-3-激酶(PI3K)、蛋白激酶B(Akt)、p-Akt及上皮间质化标志物钙黏蛋白E(E-cad)、闭锁小带蛋白1(ZO-1)、F-肌动蛋白(F-actin)、α-平滑肌肌动蛋白(α-SMA)、纤连蛋白1(FN-1)和vimentin蛋白表达。利用靶基因预测库预测miR-155靶基因,荧光素酶报告载体鉴定靶基因。结果培养后48 h,对照组细胞状态良好,细胞间连接紧密,形状规则。TGF-β2组细胞呈较明显的梭形或纺锤体形状,多数细胞表现为纤维状,细胞排列松散。TGF-β2组细胞miR-155相对表达量为0.92±0.14,明显高于对照组的0.35±0.06,差异有统计学意义(t=7.242,P=0.003);miR-155抑制剂组细胞miR-155相对表达量为0.21±0.03,明显低于miR-155阴性对照组的0.98±0.09,差异有统计学意义(t=12.421,P<0.01)。TGF-β2组细胞迁移率明显高于对照组,穿过基底膜的细胞数明显多于对照组;miR-155阴性对照组细胞迁移率明显高于miR-155抑制剂组,穿过基底膜的细胞数明显多于miR-155抑制剂组,差异均有统计学意义(均P<0.01)。与对照组比较,TGF-β2组细胞PTEN蛋白相对表达量降低,PI3K相对表达量升高,p-Akt/Akt比值升高,上皮细胞标志蛋白E-cad、ZO-1、F-actin相对表达量降低,间质细胞标志蛋白α-SMA、FN-1、vimentin相对表达量�Objective To investigate the effect of microRNA-155(miR-155)on transforming growth factorβ2(TGF-β2)-induced epithelial-mesenchymal transition of human retinal pigment epithelial cells and its mechanism.Methods The retinal pigment epithelial cell ARPE-19 cell line was used as the research object.The cells cultured with DMEM medium were served as the control group and the cells cultured with DMEM medium containing 10 ng/ml TGF-β2 were served as the TGF-β2 group.The ARPE-19 cells transfected with miR-155 inhibitor were set as the miR-155 inhibitor group and the ARPE-19 cells transfected with miR-155 negative control were set as the miR-155 negative control group,and the cells in the two groups were cultured in DMEM medium containing 10 ng/ml TGF-β2.After 48 hours cell culture,reverse transcription-PCR was used to detect the expression of miR-155 in each group,and scratch migration test and Transwell chamber test were used to detect cell migration and invasion ability,and Western blot was used to detect the expressions of phosphate and tension homology deleted on chromosome ten gene(PTEN),phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),p-Akt and epithelial mesenchymal markers E-cadherin(E-cad),zonula occludens protein 1(ZO-1),F-actin,α-smooth muscle actin(α-SMA),fibronectin 1(FN-l)vimentin,proteins.The target gene prediction library predicted miR-155 target gene and fluorescein enzyme reporter vectors were used to identify target genes.Results After 48 hours of culture,the cells in the control group were in good condition with tight adherence and regular shape.The cells in the TGF-β2 group showed more obvious spindle shape with loose arrangement,and most of the cells were fibrous.The relative expression level of miR-155 in the cells of TGF-β2 group was 0.92±0.14,which was significantly higher than 0.35±0.06 of the control group(t=7.242,P=0.003).The relative expression level of miR-155 in the cells of miR-155 inhibitor group was 0.21±0.03,which was significantly lower than 0.98±0.09 of the miR-1

关 键 词：微小RNA-155 人视网膜色素上皮细胞 上皮-间质转化 PTEN 

分 类 号：R774.1[医药卫生—眼科]