小干扰RNA-Pax6对人晶状体上皮细胞生物学行为和上皮-间质转化的抑制作用

Inhibitory effect of siRNA-Pax6 on biological behavior and epithelial-mesenchymal transition of human lens epithelial cells

目的探讨沉默人类同源配对盒基因6(Pax6)对人晶状体上皮细胞(LECs)的生物学行为和上皮-间质转化(EMT)的作用。方法将SRA01/04人LECs分为小干扰RNA-Pax6(siRNA-Pax6)组和siRNA阴性对照(siRNA-NC)组,siRNA-Pax6组细胞转染siRNA-Pax6,siRNA-NC组细胞转染无序siRNA。转染后24、48、72 h采用细胞计数试剂盒8法检测细胞存活率;转染后48 h,采用流式细胞术检测细胞凋亡比例和不同细胞周期细胞比例;转染后24 h,采用细胞划痕实验检测细胞迁移率;转染后48 h,采用实时荧光定量PCR检测细胞内Pax6、α-晶状体蛋白A(CRYAA)、α-晶状体蛋白B(CRYAB)、转录因子Sox2及EMT相关分子平滑肌肌动蛋白(α-SMA)、钙黏附蛋白E(E-cadherin)mRNA相对表达量,采用Western blot法检测各组细胞中Pax6蛋白相对表达量。结果2个组转染后不同时间点细胞存活率总体比较差异均有统计学意义(F_(分组)=4.776,P<0.05;F_(时间)=13.535,P<0.05),其中转染后48 h和72 h,siRNA-Pax6组细胞存活率均明显低于siRNA-NC组,差异均有统计学意义(均P<0.05)。与siRNA-NC组比较,siRNA-Pax6组G_(0)/G_(1)期细胞比例明显增加,S期细胞比例明显减少,差异均有统计学意义(t=9.971、-5.063,均P<0.05)。siRNA-Pax6组细胞迁移率为(19.73±6.07)%,低于siRNA-NC组的(70.56±2.97)%,差异有统计学意义(t=-7.245,P<0.05)。siRNA-Pax6组细胞中Sox2和α-SMA mRNA相对表达量低于siRNA-NC组,E-cadherin mRNA相对表达量高于siRNA-NC组,差异均有统计学意义(t=-23.254、-5.294、6.062,均P<0.01)。siRNA-Pax6组细胞中CRYAA和CRYAB mRNA相对表达量明显高于siRNA-NC组,差异均有统计学意义(t=5.521、8.270,均P<0.01)。siRNA-Pax6组细胞中Pax6 mRNA相对表达量为0.27±0.01,低于siRNA-NC组的1.00±0.05,差异有统计学意义(t=-14.456,P<0.001);siRNA-Pax6组细胞中Pax6蛋白相对表达量为0.24±0.05,低于siRNA-NC组的1.14±0.10,差异有统计学意义(t=-4.458,P<0.001)。结论沉默Pax6可抑制人LECs的增生及EMT过程,维持人LECs内正常晶状体蛋白的表达。

Objective To explore the effect of knockdown of the homeobox gene paired-box 6(Pax6)on the biological behavior and epithelial-mesenchymal transition(EMT)of human lens epithelial cells(LECs).Methods The SRA01/04 human LECs were divided into small interfering RNA-Pax6(siRNA-Pax6)group transfected with siRNA-Pax6 and siRNA negative control(siRNA-NC)group transfected with disordered siRNA.Cell survival rate was detected by cell counting kit-8 method at 24,48 and 72 hours after transfection.Cell cycle distribution and apoptosis were analyzed by flow cytometry at 48 hours after transfection.Migratory capability of cells was examined by cell scratch test at 24 hours after transfection.The mRNA relative expression levels of Pax6,α-crystallin A(CRYAA),α-crystallin B(CRYAB),Sox2,α-smooth muscle actin(α-SMA)and E-cadherin were detected by quantitative real-time PCR at 48 hours after transfection.The relative expression of Pax6 protein was detected by Western blot at 48 hours after transfection.Results There was a significant difference in cell survival rates at different time points between the two groups(F_(group)=4.776,P<0.05;F_(time)=13.535,P<0.05).The cell survival rate of siRNA-Pax6 group was obviously lower than that of siRNA-NC group at 48 and 72 hours after transfection,and the differences were statistically significant(both at P<0.05).Compared with siRNA-NC group,the proportion of cells in G_(0)/G_(1) phase was significantly increased and the proportion of cells in S phase was significantly reduced in siRNA-Pax6 group(t=9.971,-5.063;both at P<0.05).The cell migration rate of siRNA-Pax6 group was(19.73±6.07)%,which was lower than(70.56±2.97)%of siRNA-NC group,showing a statistically significant difference(t=-7.245,P<0.05).The relative expressions of Sox2 mRNA andα-SMA mRNA were lower,and the relative expression of E-cadherin mRNA was higher in siRNA-Pax6 group than siRNA-NC group,with statistically significant differences between them(t=-23.254,-5.294,6.062;all at P<0.01).The relative expression of CRYAA mRNA and CRYAB mRNA was significantly higher in siRNA-Pax6 group than siRNA-NC group,and the differences were statistically significant(t=5.521,8.270;both at P<0.01).The relative expressions of Pax6 mRNA and protein in siRNA-Pax6 group were 0.27±0.01 and 0.24±0.05,respectively,which were both lower than 1.00±0.05 and 1.14±0.10 in siRNA-NC group,showing statistically significant differences(t=-14.456,-4.458;both at P<0.001).Conclusions Silence of Pax6 can suppress the proliferation and EMT of human LECs and enhance the expression of crystallin.