沉默信息调节因子1SIRT1基因沉默诱导肝癌细胞老化及机制研究.doc

沉默信息调节因子1（SIRT1）基因沉默诱导肝癌细胞老化及机制研究 宋春丽1，任吉华1，张祯祯2，陶颖1，陈娟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 重庆市 400016 重庆医科大学附属儿童医院感染科 重庆市 400014 [摘要] 目的 探讨慢病毒介导的SIRT1基因沉默对肝癌细胞老化的影响及机制研究。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Western blot验证SIRT1基因的沉默效果。台盼蓝排斥实验检测细胞生长情况；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的表达并且同时抑制肝癌细胞的增殖。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加（P0.05），并伴随衰老相关基因p53和p21蛋白表达增加。 结论SIRT1的沉默诱导了细胞老化，并同时促进p53和p21蛋白的表达，提示SIRT1基因沉默可能通过p53/p21途径促进细胞衰老。 [关键词]? SIRT1；肝癌；老化 [中图分类号]?R34 Gene silencing of SIRT1 inhibits proliferation and induces Senescence in hepatocelluar carcinoma cells Song Chunli1 Ren Jihua1 Zhang Zhenzhen2 Tao Ying1 Chen Juan1 Key Laboratory of Molecular Infectious Diseases,Ministry of Education,Chongqing Medical University,Chongqing,400016,China Department of Infectious Diseases,Childrens Hospital,Chongqing Medical University,Chongqing,400014 【Abstract】 Objective To investigate the effect of SIRT1 silencing on cellular senescence in Hepatocellular carcinoma cells(HCC) and the mechanism. Methods SIRT1 expression was downregulated by lentivirus expressing shRNA targeting SIRT1 and SIRT1 protein level was measured by western blot. Cell proliferation in response to SIRT1-silencing was determined by trypan blue exclusion assay.The senescence was observed by SA-β-gal staining and senescence-associated proteins p53 and p21 were further analyzed by western blot. Results Lentivirus expressing shRNA targeting SIRT1 could significantly knockdown and SIRT1 expression as measured by western blot. SIRT1 knockdown resulted in a decrease in proliferation and increased staining of Senescence-associated β-galactosidase (p0.05). Western blot further fount that gene silencing of SIRT1 induced expression of senesence-associated proteins p53 and p21.Conclusion SIRT1 silencing induced cellular senescence and expression of senesence-associated proteins p53 and p21. Our finding suggested that SIRT1 silencing induced cellular senescence ,a