Authors: Tian Fang, Zhao Jinzhi, Teng He, Huang Liangyu, Liu Xun, Su Ruihong, Gao Meizi, Zhang Xiaomin, Li Xiaorong, Dong Lijie, Zhang Hong
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.
HLECs (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.
Cultured 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).
KLF6 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.