野生型p53对肝癌细胞POLD1基因表达及细胞恶性表型的影响  

Effect of overexpression of wild-type p53 on POLD1 expression and malignant cell behavior in human hepatocellular carcinoma cell line SMMC-7721

作　　者：韦长元[1] 刘起理[1] 廖柳凤[1] 徐恒[2] 谭晓虹[1] 

机构地区：[1]广西医科大学附属肿瘤医院,广西壮族自治区南宁市530021 [2]广西医科大学医学实验中心,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2011年第23期2443-2449,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.303296~~

摘　　要：目的:探讨野生型p53对人肝癌细胞SMMC-7721细胞增殖和细胞恶性表型影响的一种可能机制.方法:设计并构建p53特异性小干扰shRNA绿色荧光真核表达质粒(p53-siRNA)和表达EGFP-p53融合蛋白的p53绿色荧光真核增强表达质粒(pEGFP-p53),通过脂质体Lipofection-2000介导转染,将表达pEGFP-p53重组质粒、p53-siRNA及空载体pEGFP-C1转染入SMMC-7721细胞;经G418筛选,获得稳定细胞系7721-p53、7721-C1;7721-p53RNAi、7721-NC.通过RT-PCR检测转染后p53、POLD1mRNA表达水平.通过生长曲线测定,克隆形成实验,了解SMMC-7721细胞在p53表达水平改变后细胞癌性的变化.结果:与人肝癌细胞SMMC-7721比较,转染质粒pEGFP-p53的野生型p53高表达组,p53mRNA表达量增高,POLD1基因的表达量降低;而转染pGPU6/GFP/neo-p53i-769的p53低表达组,p53mRNA表达量降低,POLD1mRNA表达量增高,其他组较空白对照无明显变化.对高表达野生型p53的人肝癌细胞系SMMC-7721-pEGFP-C1-p53,低表达野生型p53的人肝癌细胞系SMMC-7721-pGPU6/GFP/neo-p53i-769,和普通人肝癌细胞SMMC-7721分别进行MTT和平板克隆形成实验.MTT结果发现:SMMC-7721-pGPU6/GFP/neo-p53i-769的细胞其生长速度比普通肝癌细胞的要快,而SMMC-7721-pEGFP-p53的细胞较普通肝癌细胞生长速度较慢.克隆形成实验结果显示:pEGFP-C1-p53、pGPU6/GFP/neo-p53i-769组克隆形成率分别为38.1%和72.6%,与普通肝癌细胞SMMC-7721的克隆形成率52.6%相比,均有统计学差异(P<0.05).结果显示:在人肝癌细胞SMMC-7721中,野生型p53高表达组能够抑制POLD1的基因转录,并抑制细胞增殖活性;而低表达组能够促进POLD1的基因转录,促进细胞增殖.结论:p53对POLD1的调控作用可能是p53对肝癌细胞增殖和细胞恶性表型影响的一种新的机制.AIM： To investigate the impact of overexpression of wild-type p53 on cell proliferation and malignant phenotype in human hepatocellular carcinoma cell line SMMC-7721 and to explore possible mechanism involved. METHODS： Enhanced tein gene-containing green fluorescence proeukaryotic expression plasmids expressing p53-specific small interfering RNA （shRNA） （p53-siRNA） or wild-type p53 （pEGFP-p53） were constructed and introduced into SMMC-7721 cells by Lipofection- 2000-mediated transfection. Meanwhile, the pEGFP-C1 empty vector was also transfected into SMMC-7721 cells. Cell lines stably expressing p53-siRNA, pEGFP-p53 or pEGFP-C1 were screened in medium containing G418. After transfection, the expression of p53 and POLD1 mRNAs was detected by RT-PCR. The changes in malignant cell behavior were determined by cell growth curve determination and colony formation assay. RESULTS： Compared to control SMMC-7721 cells, p53 mRNA expression was increased and POLD1 gene expression was decreased in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene, while p53 mRNA expression was reduced and POLD1 mRNA expression was increased in SMMC-7721 cells transfected with the plasmid carrying p53- siRNA. MTT results showed that cell growth rate was faster in SMMC-7721 cells transfected with the plasmid carrying p53-siRNA than in control SMMC-7721 cells, but was slower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells. Colony formation assay showed that colony formation rate was lower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells （38.1% vs 52.6%, P 〈 0.05）, but was higher in cells tranfected with the plasmid carrying p53-siRNA than in control cells （72.6% vs 52.6%, P 〈 0.05）. High expression of wild-type p53 inhibited POLD1 transcription and cell proliferation, while low expression of wild-type p53 promoted POLD1 transcription and cell proliferation. CONCLUSION： Wild-type p53 controls li

关 键 词：SMMC-7721 POLD1 P53 RNA干扰 

分 类 号：R735.7[医药卫生—肿瘤]