Kuippel样因子6经活化转录因子4通路对晶状体上皮细胞凋亡的调控作用

Regulation of Kriippei-like factor 6 via activating transcription factor 4 pathway to apoptosis of human lens epithelial cells

目的以活化转录因子4（ATF4）为靶点，探讨Krüppel样因子6（KLF6）调控紫外线B（UVB）诱导的晶状体上皮细胞（HLECs）凋亡的分子机制。方法HLECs系（HLE-B3）进行常规培养，采用脂质体转染法将构建的真核表达质粒pEGFP-C2-ATF4转染HLECs作为UVB＋ATF4转染组，并用能量密度为20 mJ/cm2的UVB照射细胞；未转染的ATF4细胞作为正常对照组。采用苏木精－伊红染色法和Hoechst染色法观察ATF4对HLECs细胞形态学的影响。将培养的细胞分为KLF6转染组及相应空质粒对照组，小干扰KLF6（siKLF6）组（转染pSilencer-KLF6质粒）及相应的空载体对照组（转染pSilencer空质粒），采用Western blot法检测细胞中ATF4蛋白的相对表达量。将培养的细胞分为4个组，联合空载体组HLECs联合转染pEGFP-C2空载体和pSilencer空载体；KLF6＋pSilencer空质粒组HLECs联合转染pEGFP-C2-KLF6和pSilencer空载体；小干扰ATF4（siATF4）＋pEGFP-C2组HLECs联合转染pEGFP-C2空载体和pSilencer-ATF4；KLF6＋siATF4组HLECs联合转染pEGFP-C2-KLF6和pSilencer-ATF4，各组细胞均暴露于UVB 200 s，采用ELISA法检测上述各组HLECs凋亡值。结果正常对照组培养的HLECs大小均匀，排列整齐，细胞核呈卵圆形，数量多且完整；UVB照射后部分细胞核固缩，细胞间隙增大，少数细胞出现核分裂；UCB＋ATF4转染组细胞数量减少，多数细胞出现核固缩和核分裂。UVB＋ATF4转染组细胞中ATF4蛋白表达条带灰度明显强于UVB＋空载体组，ATF4蛋白相对表达量分别为0.99±0.06和0.13±0.02，差异有统计学意义（t＝23.13，P〈0.01）。KLF6转染组细胞中KLF6和ATF4蛋白相对表达量均明显高于其空载体对照组，siKLF6组细胞中KLF6和ATF4蛋白相对表达量均明显低于其空载体对照组，差异均有统计学意义（均P〈0.01）。ELISA检测显示ATF4转染组HLECs凋亡值为1.37±0.11，明显高于正常对照组的0.31±0.11，差异有统计学意义（t＝8.034，P＝0.001）；KLF6＋pSilencer空质粒组细胞凋亡值明显高于联合空载体组，siATF4＋pEGFP-C2组细胞凋亡值明显低于联合空载体组，差异均有统计学意义（P〈0.01，P＝0.02）；KLF6＋siATF4组细胞凋亡率明显低于KLF6＋pSilencer空质粒组，差异有统计学意义（P〈0.01）。 结论KLF6通过活化ATF4促进UVB诱导的HLECs凋亡，ATF4基因沉默可使细胞凋亡值明显降低，因此ATF4可作为KLF6调控HLECs凋亡的靶因子，也是KLF6调控LECs凋亡的主要生物机制之一。

Objective To investigate the regulating effects of Krüppel-like factor 6 （KLF6） on the apoptosis of human lens epithelial cells （HLECs） by activating transcription factor 4 （ATF4） pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.MethodsHLECs （HLE-B3） were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group, and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B （UVB） for 200 seconds, The morphological changes of the cells were observed by hematoxylin ＆ eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 （siKLF6） and pSilencer plasmid, respectively, and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group; pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6＋ pSilencer group; pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4＋ pEGFP-C2 group; pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6＋ siATF4 group, and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay. ResultsCultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis, karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group, the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB＋ ATF4 transfected group and UVB＋ pEGFP-C2 plasmid group, respectively, with a significant difference between them （t=23.13, P〈0.01）. The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group, and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group （all at P〈0.01）. ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37±0.11, which was significantly higher than 0.31±0.11 in the normal control group （t=8.034, P=0.001）; the apoptotic rate of the cells was increased in the KLF6＋ pSilencer group and decreased in the siATF4＋ pEGFP-C2 group in comparison with the empty plasmid group （P〈0.01, P=0.02）. In addition, the apoptotic rate in the KLF6＋ siATF4 group was remarkably lower than that in the KLF6＋ pSilencer group （P〈0.01）. ConclusionsKLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating pathway of KLF6 to apoptosis.