结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响

Effect of p53 with R213 mutation on the expression of p21 in colorectal cancer cells HCT116

目的确定在结肠癌细胞系中,p53的213位精氨酸突变后对其下游靶基因p21表达的影响。方法在p53缺失的结肠癌细胞系HCT116^(-/-)中转染p53的R213位突变的质粒;荧光素酶双报告基因系统检测p53靶基因启动子活性;EMSA检测p53与其靶基因p21的结合能力;real-time PCR和Western blot检测p53下游靶基因p21的表达。结果 p53蛋白213位精氨酸突变为赖氨酸后,明显降低了p53靶基因的启动子活性(P<0.05);与靶基因p21启动子的结合能力明显降低(P<0.05);并抑制了p21基因的表达(P<0.05)。而213位突变为谷氨酰胺后,迁移率比突变前明显变快。结论在人的结肠癌细胞中,p53蛋白R213位的不同突变对下游靶基因p21有不同的影响,提示存在着不同的调控机制。

Objective To determine impacts of p53 with R213 mutation on the expression of its downstream target gene p21 in HCT116^-/- colorectal cancer cell line. Methods Mutations of p53 at R213Q （R/Q） and R213K （R/K） were respectively constructed and transfected into the p53 null HCT116 cells. The promoter activity drived by p53 was detected by dual-luciferase reporter system. EMSA assay was used to detect the ability of p53 binding to the promoter of its target gene p21. The expression of p53 downstream target genes p21 was examined by real-time PCR and Western blot experiments. Results The mutation of R213K disrupted the efficiency of p53 transactivation （P 〈 0. 05 ）. The mutants were less efficient in targeting the consensus binding sequences of p53 in the regulatory region of p21 gene（ P 〈 0.05 ）, In addition, The R/K mutant attenuated the expression of p21 gene （ P 〈 0. 05 ）. The mobility of R213Q was increased. Conclusions The different mutations of Arginine 213 have different impact on p21 in colorectal cancer cells. It could exist diverse regulating mechanisms.