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.

Keywords

genetic imprinting / gene PEG10 / L0₂ hepatocytes / hydrogen peroxide / apoptosis

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