14-3-3σ对p73基因转录活性的影响

Effect of 14-3-3σ on Transcriptional Activity of p73 Gene

背景与目的:p53基因是14-3-3σ的主要调节基因,活化的p53可以诱导14-3-3σ的表达,反过来14-3-3σ又可以稳定p53的表达并且增加其转录活性。p53基因家族中的其它成员p63和p73也存在着一些与p53基因相似的功能。本研究目的在于探讨14-3-3σ对p73基因转录活性的影响。方法:采用荧光素酶报告剂分析、反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)及Western blot研究在p53缺失型人类肺癌细胞系H1299中14-3-3σ对p73基因转录活性的调节作用;用集落形成实验研究在p53突变型人类乳腺癌细胞系MDA-MB-436中14-3-3σ对p73基因转录活性的调节作用。结果:荧光素酶报告剂分析结果显示,在H1299细胞系中,转染p73可以使bax和p21WAF1启动子介导的荧光素酶表达增加;共转染p73和14-3-3σ后,bax和p21WAF1启动子介导的荧光素酶表达均比单独转染p73时的表达高,并且荧光素酶的表达和14-3-3σ的转染剂量之间存在着浓度依赖性(P<0.01)。RT-PCR和Western blot结果也显示,转染p73可以使bax和p21WAF1的表达增加;共转染p73和14-3-3σ后,bax和p21WAF1的表达均比单独转染p73时的表达高(P<0.01)。集落形成实验结果显示,在MDA-MB-436细胞系中,转染p73可以使细胞的克隆数减少;共转染p73和14-3-3σ后,细胞克隆数比单独转染p73时少(P<0.01)。结论:14-3-3σ可以增加p73基因的转录活性,并且14-3-3σ对p73基因转录活性的调节存在剂量依赖性。

BACKGROUND ＆ OBJECTIVE： p53 gene is the main regulator of 14-3-3σ. Activated p53 could induce the expression of 14-3-3σ, while 14-3-3σ stabilizes the expression of p53 and enhances its transcriptional activity, p63 and p73, the members of p53 family, also have some functions similar to p53. This study was to investigate the effect of 14-3-3σ on the transcriptional activity of p73. METHODS： Luciferase reporter assay, reverse transcription-polymerase chain reaction （RT-PCR）, and Western blot were used to evaluate the effect of 14-3-3σ on the transcriptional activity of p73 in p53-deficient human lung carcinoma cell line H1299. Colony formation test was used to evaluate the effect of 14-3-3σ on the transcriptional activity of p73 in p53-mutant human breast cancer cell line MDA-MB-436. RESULTS; The luciferase activities induced by bax and p21^WAF1 promotors were significantly higher in p73-transfected H1299 cells than in control H1299 cells （P〈0.01）, and were further increased by the transfection of p73 （25 ng） and 14-3-3σ （100, 200, and 400 ng） in a dose-dependent manner （P〈0.01）. The expression of bax and p21^WAF1 were higher in p73-transfected H1299 cells than in control H1299 cells, and were significantly higher in p73- and 14-3-3σ-transfected H1299 cells than in p73-transfected H1299 cells （P〈0.01）. The number of colonies was fewer in p73-transfected MDA-MB-436 cells than in control MDA-MB-436 cells, and the colonies were significantly smaller in p73- and 14-3-3σ-transfected H1299 cells than in p73-transfected H1299 cells （P〈0.01）. CONCLUSION： 14-3-3σ can enhance the transcriptional activity of p73 in a dose-dependent manner.