SIRT1基因沉默诱导肝癌细胞老化及其机制  

Silent information regulator 1 gene induces aging of hepatocellular carcinoma cells via p53/p21 pathway

作　　者：宋春丽[1] 任吉华[1] 张祯祯[2] 陶颖[1] 陈娟[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016 [2]重庆医科大学附属儿童医院感染科,重庆400014

出　　处：《第三军医大学学报》2012年第19期1929-1932,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(81171628)~~

摘　　要：目的探讨慢病毒介导的沉默信息调节因子1(silent information regulator1,SIRT1)基因沉默对肝癌细胞老化的影响及机制。方法通过慢病毒介导的shRNA干扰技术靶向沉默SIRT1的表达,并通过Real-time PCR和Westernblot验证SIRT1基因的沉默效果。BrdU标记实验检测细胞增殖情况,流式细胞仪检测细胞周期变化;β-半乳糖苷酶染色观察肝癌细胞有无老化;Western blot检测衰老相关基因p53和p21蛋白的变化。结果慢病毒介导的shRNA能显著抑制细胞SIRT1的mRNA及蛋白水平。SIRT1沉默能抑制肝癌细胞的增殖,抑制率达70%～80%;同时,SIRT1沉默能显著诱导G1期细胞周期阻滞:感染shCont的细胞处于G1期比例为(44.29±0.36)%,而感染shSIRT1-1的细胞处于G1期细胞为(69.20±1.24)%,感染shSIRT1-2的细胞处于G1期比例为(65.86±1.75)%。SIRT1沉默后肝癌细胞中衰老相关的β-半乳糖苷酶染色显著增高,阳性细胞占40%～50%(P<0.05),并伴随衰老相关基因p53和p21蛋白表达增加。结论 SIRT1基因沉默可能通过p53/p21途径促进细胞衰老。Objective To study the effect of lentivirus-mediated silent information regulator 1 gene （ SIRT1 ） on aging of hepatocellular carcinoma cells （HCCs） and its mechanism. Methods SIRT1 expression was targeted by lentivirus-mediated shRNA interference technology. Effect of SIRT1 on aging of HCCs was detected by RT-PCR and Western blot analysis, respectively. Proliferation of HCCs was assayed by BrdU- labeling test. Cell cycle of HCCs was displayed by flow cytometry. Aging of HCCs was observed with SA-13-gal staining. Aging-related p53 and p21 proteins were demonstrated by Western blot analysis. Results Lentivirus- mediated shRNA significantly down-regulated the SIRT1 mRNA and protein expression levels in HCCs. SIRT1 suppressed the proliferation of HCCs with a suppression rate of 70% - 80%, and significantly induced cell cycle arrest at stage G1. The proportion of shCont-, SIRT1- and SIRT2-infected HCCs at stage G1 was about （42.29±0.36 ） %, （69. 20±1.24 ） %, and （65.86±1.75 ） %, respectively. The aging-related 13-galactosi- dase-stained HCCs were significantly greater with a positive rate of 40% - 50% （P 〈 0.05）, and the aging- related p53 and p21 protein expression levels were significantly higher. Conclusion SIRT1 promotes the aging of HCCs possibly through the p53/p21 pathway.

关 键 词：沉默信息调节因子1 肝细胞癌 肿瘤细胞系 细胞衰老 

分 类 号：R394.2[医药卫生—医学遗传学] R73-354[医药卫生—基础医学]