摘要
目的探讨甲基化CpG集合蛋白2(MeCP2)对晶状体上皮细胞(LECs)生物学行为和上皮-间质转化(EMT)发生的作用及其可能机制。方法根据细胞中转染物序列不同将人晶状体上皮细胞株SRA01/04分为MeCP2类似物组、MeCP2-空质粒组和小干扰RNA-MeCp2(si-MeCP2)组,分别将相应序列的质粒转染SRA01/04细胞。转染后24 h采用逆转录PCR法检测各组细胞中MeCP2 mRNA相对表达量;于转染后48 h采用划痕试验检测细胞迁移率;采用免疫荧光染色测定细胞内Wnt3a蛋白表达;采用Western blot法检测细胞内β-catenin、钙黏附蛋白-E(E-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶(MMP)-9、MMP-7和分泌型卷曲相关蛋白5(SFRP5)蛋白表达。结果转染后24 h,MeCP2-拟似物组、MeCP2-空质粒组和si-MeCP2组细胞中MeCP2 mRNA相对表达量总体比较,差异有统计学意义(F=4773.00,P<0.001)。划痕试验结果显示,MeCP2-拟似物组、MeCP2-空质粒组和si-MeCP2组细胞迁移率分别为(57.45±5.20)%、(32.71±10.02)%和(17.77±9.22)%,总体比较差异有统计学意义(F=124.00,P<0.001),其中si-MeCP2组细胞迁移率明显低于MeCP2-拟似物组和MeCP2-空质粒组,差异均有统计学意义(均P<0.001)。MeCP2-拟似物组、MeCP2-空质粒组和si-MeCP2组细胞中Wnt3a蛋白荧光强度(A值)分别为75.92±6.10、52.03±5.22和28.75±3.39,总体比较差异有统计学意义(F=221.30,P<0.001),其中MeCP2-mimics组显著高于MeCP2-CN组,si-MeCP2组细胞中Wnt3a蛋白荧光强度明显低于MeCP2-空质粒组和MeCP2-拟似物组,差异均有统计学意义(均P<0.001)。si-MeCP2组细胞内E-cadherin蛋白相对表达量明显高于MeCP2-拟似物组和MeCP2-空质粒组,细胞中β-catenin、Vimentin、MMP-9和MMP-7蛋白相对表达量明显低于MeCP2-拟似物组和MeCP2-空质粒组,差异有统计学意义(均P<0.01)。MeCP2-拟似物组、MeCP2-空质粒组和si-MeCP2组细胞内SFRP5蛋白相对表达量分别为27.19±0.03、47.54±0.05和74.93±0.05,总体比较差异有统计学意义(F=183.49,P<0.001),其中si-MeCP2组细胞内SFRP5蛋白相对表达量明显高于MeCP2-拟似物组和MeCP2-空质粒组,差异均有统计学意义(均P<0.001)。结论MeCP2能刺激人LECs发生EMT,其作用机制可能与其靶向抑制SFRP5表达,继而激活Wnt3a/β-catenin信号通路有关。
Objective To investigate the role of methyl-CpG-binding protein 2(MeCP2)in the regulation and epithelial-mesenchymal transition(EMT)of human lens epithelial cells(LECs)and its possible mechanism.Methods Human LEC lines(SRA01/04)were divided into MeCP2-mimic group,MeCP2-NC group and small interferening RNA-MeCP2(si-MeCP2)group,and MeCP2 analog plasmid,blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection,the migration rate of the cells was evaluated by scratching test,and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions ofβ-catenin,E-cadherin,Vimentin,matrix metallo proteinase(MMP)-9,MMP-7 and secreted frizzled-related protein 5(SFRP5)proteins in the cells were detected by Western blot.Results After 24 hours of transfection,the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group(F=4773.00,P<0.001).The migrating rate of the cells in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was(57.45±5.20)%,(32.71±10.02)%and(17.77±9.22)%,respectively,showing a significant difference among the three groups(F=124.00,P<0.001),and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group(both at P<0.001).The relative expressing intensity(absorbance)of Wnt3a in the cells of the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 75.92±6.10,52.03±5.22 and 28.75±3.39,respectively,with a significant difference among three the groups(F=221.30,P<0.001),and the relative expressing intensity(absorbance)of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group(both at P<0.001).The relative expressing level of E-cadherin protein was significantly elevated and the expressions ofβ-catenin,Vimentin,MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group(all at P<0.01).The relative expressing level of SFRP5 protein in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 27.19±0.03,47.54±0.05 and 74.93±0.05,respectively,showing a statistical difference among the three groups(F=183.49,P<0.001),and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group(both at P<0.001).Conclusions MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.
作者
牛超
吴众
黄亚琳
张颖
王应飞
李晓华
陆文龙
Niu Chao;Wu Zhong;Huang Yalin;Zhang Ying;Wang Yingfei;Li Xiaohua;Lu Wenlong(Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,Henan Key Laboratory of Ophthalmology and Visual Sciences,People's Hospital of Zhengzhou University,People's Hospital of Henan University,Zhengzhou 450003,China;School of Medical Techndoqy and Engineering,Henan University of Science and Technology,Luoyang 471000,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2020年第1期32-37,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目(81770952)
河南省自然科学基金面上项目(162300410296)。
作者简介
通信作者:李晓华,Email:yksbls2390@126.com。
