P21对胃癌细胞MGC-803的增殖抑制及对POLD1基因表达的调控

P21 suppresses cell proliferation and downregulates POLD1 expression in human gastric cancer cell line MGC-803

目的:研究P21对POLD1基因的调控通路,以探索阻断癌细胞恶性增殖的机制.方法:实验主要分3组:阴性对照组(转染空载体pXJ41-neo的803-pXJ细胞)、空白对照组(胃癌细胞MGC-803)、实验组(转染P21重组真核表达质粒pXJ41-p21的803-p21细胞).MTT实验分析细胞增殖变化,流式细胞仪检测细胞凋亡水平,实时荧光定量PCR技术检测基因表达水平的变化,Westernblot分析蛋白表达差异.结果:MTT实验显示,与空白对照和阴性对照组相比,实验组细胞增殖受到明显抑制,凋亡率(%)增高(11.36±0.51vs7.39±0.17,7.69±0.47,F=85.338,均P<0.05).实验组P21mRNA表达水平显著提高(2.15±0.23vs1.05±0.11,1.00±0.00,F=59.054,均P<0.05),POLD1则显著下调(0.45±0.07vs1.09±0.13,1.00±0.00,F=49.907,均P<0.05);P21、P125蛋白表达变化与基因变化相一致.cyclinE、Rb1基因表达均上调,CDK2基因表达下调,c-myc基因表达则变化不大.结论:P21抑制了胃癌细胞的增殖、促进其凋亡,同时抑制了POLD1基因的表达,这种抑制作用可能通过CDK2、cyclinE、Rb1等细胞周期因子实现.

AIM：To investigate whether and how P21 regulates POLD1 expression in human gastric cancer cell line MGC-803 and to find new clues to blocking the malignant proliferation of cancer cells. METHODS：MGC-803 cells were divided into three groups：blank control group（untransfected cells）,negative control group（cells transfected with an empty vector pXJ41-neo）,and experimental group（cells transfected with a eukaryotic expression plasmid pXJ41-p21）.After transfection,cell proliferation was detected by MTT assay;cell apoptosis was detected by flow cytometry;and the mRNA and protein expression was detected by quantitative real-time PCR and Western blot,respectively. RESULTS：Compared to the two control groups, cell proliferation was significantly inhibited,cell apoptosis was increased（11.36±0.51 vs 7.39± 0.17,7.69±0.47,F=85.338,P0.05）,and the mRNA and protein expression of POLD1 was inhibited in the experimental group.In addition,the relative expression levels of cyclin E and Rb1 increased,that of CDK2 decreased,and that of c-myc showed little change in the experimental group when compared to the two control groups. CONCLUSION：P21 can suppress cell proliferation and promote apoptosis in human gastric cancer cell line MGC-803.P21 can also suppress POLD1 expression possibly by regulating the expression of CDK2,cyclin E,Rb1 or other cell cycle factors in MGC-803 cells.