目的探讨小干扰RNA-Yes相关蛋白1(siRNA-YAP1)对转化生长因子β 2(TGF-β 2)诱导的人晶状体上皮细胞(LECs)发生上皮-间质转化(EMT)的抑制作用。
方法将人LECs株HLEB-3细胞分为对照组和TGF-β 2诱导组，正常对照组采用无血清低糖培养基处理细胞，TGF-β 2诱导组采用10 ng/ml TGF-β 2处理细胞，分别培养24 h。另将HLEB-3细胞分为siRNA空载体组、siRNA-YAP1转染组、siRNA空载体+TGF-β 2组和siRNA-YAP1+TGF-β 2组，分别采用含有siRNA空载体和siRNA-YAP1序列的质粒转染细胞。采用实时荧光定量PCR、细胞免疫荧光染色和Western blot法检测各组细胞中转录共激活因子Yes相关蛋白1(YAP1)mRNA及其蛋白的相对表达量；采用细胞免疫荧光、Western blot法检测各组中LECs EMT相关标志物E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)在细胞中的表达变化。
结果TGF-β 2诱导组中E-cadherin蛋白相对表达量为0.830±0.104，低于正常对照组的1.180±0.118，TGF-β 2诱导组中Vimentin蛋白相对表达量为1.240±0.110，高于正常对照组的0.797±0.110，差异均有统计学意义( t＝3.857， P＝0.018； t＝-4.933， P＝0.008)；TGF-β 2诱导组YAP1 mRNA和蛋白的相对表达量分别为2.200±0.193和1.203±0.121，显著高于正常对照组的1.136±0.123和0.967±0.025，差异均有统计学意义( t＝-9.288， P<0.01； t＝-3.329， P＝0.029)。与siRNA空载体组比较，siRNA-YAP1转染组细胞中YAP1 mRNA及蛋白相对表达量明显下降，差异均有统计学意义(均 P<0.01)。与siRNA空载体+TGF-β 2组比较，siRNA-YAP1+TGF-β 2组细胞中E-cadherin蛋白相对表达量明显升高，Vimentin蛋白相对表达量明显下降，差异均有统计学意义(均 P<0.01)。
结论YAP1参与TGF-β 2诱导的HLEB-3细胞EMT过程，siRNA-YAP1抑制TGF-β 2诱导的人LECs EMT过程。
ObjectiveTo investigate the inhibitory effect of small interfering RNA-Yes-associated protein 1 (siRNA-YAP1) on epithelial-mesenchymal transition (EMT) in human lens epithelial cells (LECs) induced by transforming growth factor-β 2 (TGF-β 2).
MethodsHuman LECs line (HLEB-3) was cultured and divided into normal control group and TGF-β 2 induced group.The cells in the normal control group were treated with serum-free low-glucose medium for 24 hours, and the cells in the TGF-β 2 induced group were treated with additional 10 ng/ml TGF-β 2 for 24 hours.The cultured HLEB-3 cells were divided into siRNA empty vector group, siRNA-YAP1 transfection group, siRNA empty vector+ TGF-β 2 group and siRNA-YAP1+ TGF-β 2 group, and the cells were transfected with plasmid including siRNA empty vector or siRNA-YAP1 sequence according to grouping.The relative expression levels of YAP1 mRNA and protein in various groups were detected and compared by quantitative real-time polymerase chain reaction (PCR), immunofluorescence and Western blot assay, respectively.The relative expression levels of EMT marker proteins (E-cadherin and Vimentin proteins) in various groups were detected by immunofluorescence and Western blot assay.
ResultsCompared with the normal control group, the expression level of E-cadherin protein was decreased (1.180±0.118 vs.0.830±0.104) and the Vimentin protein was increased (0.797±0.110 vs.1.240±0.110) in the TGF-β 2 induced group, with significant differences between the two groups ( t=3.857, P=0.018; t=-4.933, P=0.008).The relative expression levels of YAP1 mRNA and protein in the TGF-β 2 induced group were significantly increased in comparison with the normal control group (2.200±0.193 vs.1.136±0.123; 1.203±0.121 vs.0.967±0.025), with significant differences between the two groups ( t=-9.288, P<0.01; t=-3.329, P=0.029).Compared with the siRNA empty vector group, the expression levels of YAP1 mRNA and protein in the siRNA-YAP1 transfection group were significantly reduced (both at P<0.01).Compared with the siRNA empty vector+ TGF-β 2 group, the relative expression level of E-cadherin protein was significantly enhanced and the expression level of Vimentin protein was significantly reduced in the siRNA-YAP1+ TGF-β 2 group (both at P<0.01).
ConclusionsYAP1 participates in the TGF-β 2 induced EMT in human LECs, and siRNA-YAP1 can suppress the EMT process.
郑柳,胡超,杨彬彬,等. siRNA-YAP1对TGF-β 2诱导的人晶状体上皮细胞上皮-间质转化的抑制作用 [J]. 中华实验眼科杂志,2021,39(04)：289-296.
DOI:10.3760/cma.j.cn115989-20191016-00447版权归中华医学会所有。
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郑柳、胡超：实验操作、论文撰写；杨彬彬、杨兴刚：数据整理、统计分析；丁芝祥：论文修改、经费支持
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