野生型p53调控结肠癌细胞代谢转换的研究

The experimental research of wild - type p53 in regulating metabolic transformation of colorectal cancer cells

目的观察野生型p53基因在结肠癌细胞系中能量代谢转换中的作用。方法利用流式细胞学方法检测p53在结肠癌细胞系HCTl16中对荧光标记的葡萄糖类似物：2-N[7-硝基苯-2-乙二酸，34羟氨基]-2．脱氧葡萄糖（2-NBDG）摄取的影响，进一步采用Westernblot、实时定量聚合酶链反应（Real．timePCR）等方法检测其可能的下游调控关键酶三磷酸腺苷（ATP）柠檬酸裂解酶（ACLY）、糖酵解的关键酶葡糖糖6-磷酸脱氢酶（G6PD）、乳酸脱氢酶（LDHA）、醛缩酶（ALDOA）及M2型丙酮酸激酶（PKM2）的蛋白及转录水平的表达变化。结果比较于p53缺失型（KO）HCT116细胞，p53野生型（WT）的HCT116细胞对葡萄糖的摄取能力下降41％（P〈0．05）。进一步利用Westernblot法检测能量代谢关键基因进行蛋白水平的比较及半定量分析后发现，相对于p53KO的HCT116细胞，调控细胞脂质代谢关键酶ACLY下调0．3984（t=7．154，P〈0．025）、糖酵解的关键酶G6PD下调0．3578（t=10．99，P〈0．叭）、LDHA下调0．2227（t=7．182，P〈0．05），证明上述基因在p53wT细胞中表达受抑制，而另外两个糖酵解关键酶ALDOA及PKM2的表达则无明显变化（P〉0．05）。进一步检测mRNA水平后发现p53wT的HCT116细胞中ACLY基因下调6．61倍（t=43．18，P〈0．01）、G6PD基因下调6．75倍（t=85．90，P〈0．01）及LDHA基因下调0．99倍（t=32．42，P〈0．01），而ALDOA及PKM2则无明显变化（P〉0．05）。结论野生型p53作为抑癌基因参与肿瘤能量代谢的调控过程，其可能通过抑制多种关键酶发挥作用。

Objective To investigate the regulatory effect of p53 in metabolic transformation of colorectal cancer cells. Methods Identify the role of p53 in the regulation of glucose consumption by Flow Cytometry analysis. Investigate the key enzyme of metabolic signal pathway through using Western blotting and real- time quantitative polymerase chain reaction （Real -time PCR） analysis. Results Relative to the knock- out （KO） p53 HCTll6 cells, the ability of glucose uptake appeared suppressed by 41 percent in wild - type （WT） p53 HCTll6 cells （ P 〈 0.05 ）. The protein expression of adenosine-triphosphate （ATP） -citrate lyase （ACLY）, glucose- 6- phosphate dehydrogenase （G6PD） and lactate dehydrogen- ase A （ LDHA）, key enzymes in metabolism, are decreased 0. 398 4, 0. 357 8 and 0. 222 7 respectively, as well in p53 WT HCTll6 cells （P 〈 0. 025 ）. The mRNA expression of ACLY and G6PD in p53 WT HCT116 cells exhibited approximately six - folds （ t = 43.18, P 〈 0. 01 ） and seven - folds （ t = 43.18, P 〈0.01 ） decreased, respectively. Besides, the mRNA expression of LDHA decreased approximately 50% in p53 WT HCT116 cells （t = 32. 42, P 〈 0. 01 ）. However, the protein and mRNA expression of al- dolase A （ ALDOA ） and pyruvate kinase M2 （ PKM2 ） between these cells shows no difference （ P 〉 0.05 ）. Conclusion Wild - type （WT） p53, as a tumor suppressor gene, may inhibit metabolic transfor- mation through several key enzymes.