人突变型CDK4真核荧光表达质粒的构建及对人肝细胞SMMC-7702中POLD1基因表达的调控

目的:构建包含突变型CDK4基因的真核绿色荧光表达载体,作为POLD1基因依赖的细胞周期复制调控的模型,研究POLD1基因相关的癌性增殖的机制,为干预细胞恶性增殖提供新的思路.方法:设计人突变型CDK4基因全长特异性引物进行P C R扩增人肝癌细胞系SMMC-7721总cDNA,以pEGFP-C1质粒为模板连接,得到重组质粒GFP-CDK4后进行测序和生物信息学比对分析;转染细胞分3组:实验组(转染突变型CDK4重组真核表达质粒GFP-CDK4),阴性对照组(转染空载体pEGFP-C1组)和空白对照组(SMMC-7702).通过MTT试验分析细胞增殖变化;实时荧光定量PCR技术检测CDK4、POLD1及细胞周期相关因子的表达量,Western blot检测蛋白表达的差异.结果:成功构建了人突变型CDK4基因真核表达质粒GFP-CDK4,转染到肝细胞SMMC-7702后使细胞表达融合绿色荧光的C D K4蛋白;S M M C-7721细胞中突变型的C D K4存在5个碱基突变,4个碱基插入,2个碱基缺失,这使得5个氨基酸序列发生了改变;与空白对照组及阴性对照组相比,实验组细胞增殖明显升高(0.826±0.08vs0.596±0.06,0.609±0.10,F=7.033,均P<0.05);实验组CDK4mRNA表达水平差异明显(1.94±0.11vs1.01±0.00,1.05±0.12,F=54.046,P<0.01),POLD1mRNA相应地升高(2.47±0.25vs1.16±0.00,1.26±0.23,F=135.496,P<0.01);稳定转染细胞的蛋白水平变化趋势与基因相同,其中实验组CDK4(0.65±0.03vs0.41±0.03,0.39±0.05,F=14.665,均P<0.05),P125(0.54±0.04vs0.30±0.07,0.25±0.06,F=11.788,均P<0.05).结论:人突变型CDK4基因的真核表达载体GFP-CDK4显著促进肝细胞的增殖能力,这与POLD1基因及P125蛋白的高表达相关.

Transfection of a eukaryotic vector expressing a mutant CDK4 up-regulates POLD1 expression in SMMC-7702 cells

AIM:To construct a eukaryotic expression vector encoding a mutant CDK4 protein and to investigate the effect of transfection of this vector on POLD1 expression in SMMC-7702 cells.METHODS:The mutant CDK4 gene was amplified by RT-PCR from total RNA isolated from the human hepatocarcinoma cell line SMMC-7721,digested,and inserted into the eukaryotic expression vector pEGFP-C1.The resultant recombinant plasmid was confirmed by sequencing.After the recombinant plasmid was transfected into SMMC-7702 cells using Lipofectamine 2000,the expression of fusion protein was observed by fluorescence microscopy,and expression of CDK4 and POLD1 mRNAs was detected by real-time PCR.RESULTS:The eukaryotic expression plasmid GFP-CDK4 was successfully constructed.The mutant CDK4 gene contained 5 base mutation sites,4 base insertions and 2 deletions,which caused 7 amino acids to change.Compared to non-tranfected cells or cells transfected with the pEGFP-C1 vector,cell proliferation was significantly higher in cells transfected with the recombinant vector(0.826 ± 0.08 vs 0.596 ± 0.06,0.609 ± 0.10,F = 7.033,P < 0.05).The expression levels of CDK4 and POLD1 genes in cells transfected with the recombinant vector was significantly higher than those in the two control groups(1.94 ± 0.11 vs 1.01 ± 0.00,1.05 ± 0.12,F = 54.046,P < 0.01;0.54 ± 0.04 vs 0.30 ± 0.07,0.25 ± 0.06,F = 11.788,P < 0.05).Similar results were also obtained for the protein expression levels of CDK4(0.65 ± 0.03 vs 0.41 ± 0.03,0.39 ± 0.05,F = 14.665,P < 0.05) and P125(0.54 ± 0.04 vs 0.30 ± 0.07,0.25 ± 0.06,F = 11.788,P < 0.05).CONCLUSION:Tranfection of the eukaryotic expression plasmid GFP-CDK4 significantly increases the proliferation and invasion of SMMC-7702 cells possibly by up-regulating POLD1 expression.