OBJECTIVE: p53 gene is the most important human tumor suppressor gene. The purpose of this study was to explore the effect of p53 and its mutants on the transcriptional regulation of RhoE. METHODS: We constructed PEGFP-wt-p53 plasmid, and then synthesized p53 mutation hotspot plasmid(single point and double point mutations) by site-directed mutagenesis PCR technology. pGL3-RhoE-promotor-Luc plasmid transiently transfected the wild-type and mutant p53 plasmid into PC3(p53-null) cells. Then the transcription activities of p53 and its mutants on RhoE were assessed by Dual-Luciferase reporter assay, and RhoE protein expression analyzed by western-blot. RESULTS: p53 protein could regulate the transcription activity of RhoE (P<0.05), but the mutants lost this function. The effect of the different p53 mutants on RhoE transcription regulation showed no significant difference (P>0.05). The results was confirmed by western-blot on protein level. CONCLUSION: RhoE is one of the p53 regulated genes, but once p53 mutated, it would not able to regulate the transcription activity of RhoE anymore.%目的:探索p53蛋白及其突变体对RhoE基因转录调控的影响.方法:构建pEGFP-wt-p53质粒,利用基因定点突变PCR技术构建p53单点和双点突变体质粒,构建pGL3-RhoE-promotor-Luc质粒.以P21基因的启动子序列(P21-promoter-luc)为阳性对照,EGFP-N1空载体为阴性对照,将野生型和突变型p53质粒瞬时转染PC3 (p53-null)细胞,利用双荧光素酶报告基因和Westernblot方法检测p53蛋白及其突变体蛋白在转录水平和蛋白水平对RhoE基因表达的影响.结果:电泳及测序表明以上质粒均构建成功.双荧光素酶报告基因检测显示野生型p53蛋白可调控RhoE基因转录,但其突变体丧失对RhoE的转录调控作用(P＜0.05),且不同位点的p53突变体蛋白之间对RhoE转录调控作用的差别无统计学意义(P＞0.05).Western blot结果与基因转录结果一致.结论:RhoE是受p53蛋白调控的基因之一,而p53突变体失去了在转录水平调控RhoE表达的作用.
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