The Protective Regulating Mechanism Of Wild P53 In HepG2 Cell

Objects:In order to observe whether the over expressing of p53 has an effect on the cell proliferation of HepG2, we construct the recombinant eukaryocyte expressing plasmid containing p53 gene, and study the protective regulating mechanism of p53 when HepG2 suffers from environment stress.Methods:Gene clone techniques was applied to construct recombinant plasmids pcDNA-GFP-p53 and pcDNA-ND1, which were then transfected into HepG2 respectively. Fluorescence staining and cell immunohistochemistry were-used to observe subcellular localization of p53 in HepG2 upon the environment stress. To analyze the protective effect of p53, following ways were operated:flow cytometry to sift apoptotic cells after staining; RT-qPCR to detect the transcriptional level of autophagy associated genes BECN1 and MAPLC3 through relative quantification; clone formation assay to watch the cell colony formation; WST-8 to evaluate the proliferation activity of HepG2 cultured with doxorubicin. How p53 regulates the mitochondrial gene mtNDl was researched by RT-qPCR and western blot. Flow cytometry, RT-qPCR, mitochondrial membrane potential staining were performed respectively with the purpose of showing the influence on HepG2 by over expression of mtND1.Results:1. We construct recombinant plasmids pcDNA-GFP-p53 and pcDNA-NDl successfully and acquire stable transfection cell lines by G418 screening.2. Fluorescence staining shows that the subcellular localization of p53 in HepG2 shifts from nucleus to cytoplasmic upon the environment stress such as oxidative stress, starve and so on.3. Compared with normal conditions, cells over expressing p53 exhibit the anti-apoptosis activity, up-regulating of autophagy associated genes BECN1 and MAPLC3, stronger colony forming ability and less sensibility to doxorubicin once under stress. While the phenomena above are reversed by adding Pft-α.4. mtNDl gene exists the DNA sequence that binding with p53 specifically. After incubation of purified p53 protein and mtND1 in vitro, the latter shows the declining migration rate in agarose gel electrophoresis.5. p53 presents bi-directional control with transcription and expression level of mtNDl between normal condition and environment stress. Over expression of mtNDl increases the degree of apoptosis, also results in the transcriptional inhibition of BECN1 and MAPLC3, losing of mitochondrial membrane potential.Conclusions:The over expression of p53 in HepG2 contributes to anti-apoptosis, maintaining prolification activity when suffering oxidative stress, starve, anticarcinogen and so on. That protective function may be connected with negative control on mitochondrial gene mtND1, of which over expression would lead to loss of mitochondrial membrane potential, and induce apoptosis easily.