Suppressive effects of genomic imprinted gene PEG10 on hydrogen peroxide-induced apoptosis in L02 cells

Abstract

The effects of PEG10 on hydrogen peroxide (H2O2)-induced apoptosis in human normal liver cell line L02 were investigated. The PEG10 gene was transfected into L02 cells by lipofectamine, the positive clone was screened by G418 and defined as L02/PEG10, while the cell transfected with empty expression vector (pEGFP-N1) was defined as L02/vector. L02/vector and parental L02 cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H2O2 (50-400 mmol/L) was administered to induce the apoptosis of L02 cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L02/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H2O2 for 24 h, the cellular growth inhibition rate of L02/PEG10 cells was significantly lower (58.2%) than that of L02 (92.5%) and L02/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L02/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L02 and L02/vector cell lines, but not in L02/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L02 by antagonizing H2O2-induced apoptosis.