SIRT1差异性调节不同p53表型肝细胞肝癌的发生机制

SIRT1 differently regulates oncogenesis by diverse p53 types in hepatocellular carcinoma

目的研究沉默信息调节因子2相关酶1（silentmating—typeinformationregulation2homologue1，Sirtuinl或SIRT1）在野生型及突变型p53肝细胞肝癌中差异性调节肿瘤形成的机制。方法对PLC5（249位点突变型r63），HepG2（野生型p53）细胞株进行shSIRT1慢病毒转染，通过Westernblot检测信号转导通路，通过细胞生长和增殖实验，集落形成实验，动物实验等检测HepG2细胞，HepG2一shSIRTI细胞，PLC5细胞和PLC5-shSIRT1细胞肿瘤形成能力。结果抑制SIRT1表达可降低HepG2（野生型p53）肝癌细胞的增殖，提高PLC5（突变型p53）肝癌细胞的增殖（f=3．595，P〈0．01）；敲除HepG2细胞中的SIRT1使得p53活化，激活p53下游蛋白p21的表达，敲除PLC5细胞中SIRT1能使乙酰化p53蛋白表达升高，但其下游蛋白p21不能被激活；在HepG2细胞中抑制SIRT1不能改变AMP依赖的蛋白激酶[adenosine5’-monophosphate（AMP）-activated protein kinase，AMPK]的功能（磷酸化AMPK蛋白），而在PLC5细胞（突变型p53）中抑制SIRT1可降低AMPK的功能（t=4．268，P〈0．叭）。结论在肝细胞肝癌中SIRT1激活后的作用及功能主要受到p53突变状态的影响。

Objective To uncierstand how SIRTi differently regulates oncogenesis in hepatocellular carcinoma （HCC） with wild type and mutant type p53. Methods HCC cell line PLC5 cells （249 site mutated p53 ）, and HepG2 cells （wild type p53 ） were infected with lentivirus containing shSIRT1. Western blotting was used for signaling pathway detection. Cell growth and proliferation assay, colony formation assay and tumor xenograft assay were performed to test the tumor growth ability of HepG2 cells, HepG2-shSIRT1 cells, PLC5 cells and PLC5-shSIRT1 cells respectively. Results SIRT1 silencing resulted in significant inhibition of cell proliferation in HepG2 cells but stimulating cell proliferation in PLC5 cells （t = 3. 595 ,P 〈 0. 01 ）. Acetylation of p53 was found in HepG2 （HepG2-shSIRT1） and p21 was up-regulated, however, in PLC5 （PLC5-shSIRT1） cells, acetylation of io53 was found but p21 was not induced despite of p53 activation. Silence of SIRT1 resulted in no change of AMPK function in HepG2 cells but a lower activity of AMPK in PLC5 cells （t = 4. 268,P 〈 0. 01 ）. Conclusions In HCC cell lines the function following SIRT1 activation is largely determined by p53 mutant status.