Authors: Tian Fang, Zhao Jinzhi, Huang Liangyu, Xu Manhong, Zhang Zhe, Teng He, Zhang Xiaomin, Li Xiaorong, Dong Lijie, Zhang Hong
To investigate the effect of the overexpression of Krüppel-like factor 6 (KLF6) towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation.
The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs, followed by the detection of KLF6 level by using Western blot, and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit; the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe.
The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P<0.05). In high KLF6 expression group, the cells were sparse, long and narrow in size and shape, and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 high-expression group.After UVB irradiation, the apoptosis value, relative bax expression, bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group, with statistically significant differences between them (all at P<0.05).
Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB, by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent.
Krüppel factor 6; Lens epithelial cells; Apoptosis; Ultra violetradiation B; Oxidative stress