| Objective:To investigate the p53 gene in wild type under different levels of gene expression in hepatoma cells and cell POLD1 malignant phenotype.Methods:Design and Construction of p53-specific small interfering shRNA eukaryotic expression plasmid of green fluorescence (p53-siRNA) and expression of EGFP-p53 fusion protein p53 enhanced green fluorescent eukaryotic expression plasmid (pEGFP-p53), by liposome Lipofection-2000-mediated ransfection, the expression of recombinant plasmid pEGFP-p53, p53-siRNA and pEGFP-C1 empty vector was transfected into SMMC-7721 cells; by G418 selection, stable cell line 7721-p53; 7721-p53RNAi.By RT-PCR detection of transfected p53, POLD1 the mRNA expression levels.The growth curve determination, colony formation assay to understand the SMMC-7721 cells after the change in the level of p53 expression in cancer cells changes.Results:1. High expression of wild-type p53 in human hepatoma cell line SMMC-7721-pEGFP-C1-p53 in, the ratio of grayscale value about p53/POLD1 was 3.24±0.61, while normal liver cells in gray value ratio was 0.97±0.110, shows high expression of wild-type p53 and p53 liver cancer cell lines was significantly higher than the gray value POLD1, both had statistically significant(P<0.001).2. Low expression of wild-type p53 in human hepatocellular carcinoma cell lines SMMC-7721-pGPU6/GFP/neo-p53i-769, the ratio of grayscale value about p53/POLD1 was 0.04±0.01, compared with normal liver cells in gray value ratio was 0.97±0.11, shows low expression of p53 wild-type p53 and hepatoma cell lines was significantly lower than POLD1 gray value, both had statistically significant(P<0.001).3. On the high expression of wild-type p53 in human hepatoma cell line SMMC-7721-pEGFP-C1-p53, low expression of wild-type p53 in human hepatocellular carcinoma cell lines SMMC-7721-pGPU6/GFP/neo-p53i-769, and ordinary hepatocellular carcinoma SMMC-7721 cells, respectively MTT and colony formation assay. MTT results showed that: SMMC-7721-pGPU6/GFP/neo-p53i-769 cells, the growth rate than normal liver cells, faster, and SMMC-7721-pEGFP-p53 cells compared with normal liver cancer cell growth slower;Colony formation assay showed that:pEGFP-C1-p53, pGPU6/GFP/neo-p53i-769 group clone formation rates were 38.15±0.33%, and 72.96±1.80%, and common liver cancer cell SMMC-7721's cloning efficiency of 50.53±0.15% compared were statistically significant (P<0.05).Conclusion:The human hepatoma cell SMMC-7721, the high expression of wild-type p53 gene can suppress POLDl gene transcription and cell proliferation. Low expression of wild-type p53 gene can promote the POLD1 gene transcription and cell proliferation. |
