去乙酰化酶1在肝癌干细胞更新中的机制研究

Mechanisms of SIRT1 in the renewal of cancer stem cells

目的探讨去乙酰化酶1(Sirtuins,SIRT1)在肝癌干细胞(cancer stem cells,CSCs)更新中的作用及相关的分子机制。方法获取肝CSCs,Western blotting和免疫荧光染色法检测CSCs和非-CSCs中SIRT1表达;包装过表达SIRT1的慢病毒,感染肝脏非-CSCs,Western blotting检测非-CSCs中SIRT1表达,克隆形成实验探讨过表达SIRT1后非-CSCs克隆形成能力,球体形成实验探讨非-CSCs的自我更新能力,裸鼠体内移植瘤实验探讨非-CSCs和过表达SIRT1的非-CSCs体内致肿瘤性;CSCs转染SIRT1特异性siRNA或NC-siRNA,或经不同浓度(2、4、6、8、12μmol/L)SIRT1特异性抑制剂VT6处理,然后检测CSCs的克隆形成能力、自我更新能力及体内致肿瘤性;Western blotting检测SOX2、c-Myc和Nanog蛋白表达;SIRT1特异性RNA或NC-siRNA与过表达SOX2的质粒共转染CSCs,克隆形成实验探讨CSCs克隆形成能力,球体形成实验探讨CSCs的自我能更新能力。结果 CSCs中SIRT1表达显著高于非-CSCs中SIRT1的表达(P<0.01)。与NC-siRNA转染组相比,si SIRT1转染组克隆形成能力与球体形成能力下均降了,且体内致肿瘤性也显著下降(P<0.01);感染过表达SIRT1的慢病毒后,非-CSCs中SIRT1表达显著高于空载体对照组,且与对照组相比,过表达SIRT1后非-CSCs克隆形成能力与球体形成能力均提高(P<0.01),且体内致肿瘤性也显著增强;Western blotting检测结果表明,敲低或抑制SIRT1表达后,SOX2、Nanog表达显著被抑制(P<0.01),而c-Myc的表达无显著变化(P<0.05);过表达SOX2显著逆转了敲低SIRT1表达对克隆形成能力和自我更新能力的抑制作用(P<0.01)。结论 SIRT1在肝脏CSCs的自我更新和致肿瘤过程中发挥重要作用,其可能为肝癌的治疗提供新的靶标。

Objective To investigate the role of sirtuins1（ SIRT1） in the renewal of cancer stem cells（ CSCs） and the related molecular mechanisms. Methods The expression of SIRT1 in CSCs and non-CSCs was detected by Western blotting and immunofluorescence staining; lentiviruses that overexpressed SIRT1 were packaged,and liver non-CSCs were infected,the expression of SIRT1 in non-CSCs was detected by Western blotting,the clonal formation ability of non-CSCs was investigated by clone formation assay,the self-renewal ability was investigated by sphere formation assay,and the tumorigenicity of CSCs was investigated by nude mice in vivo; CSCs were transfected with SIRT1-specific siRNA or NC-siRNA,or treated with different concentrations（ 2,4,6,8,12 μmol/L） SIRT1-specific inhibitor VT6,than the clone formation ability,self-renewal ability and tumorigenicity were determined,and the expressions of SOX2,c-Myc and Nanog were detected by Western blotting,respectively; SIRT1-specific siRNA,NC-siRNA and plasmid overexpressing SOX2 were co-transfected into CSCs,then clone formation ability was investigated by clone formation assay and the self-renewal ability was investigated by sphere formation assay. Results The expression of SIRT1 in CSCs was significantly higher than that in non-CSCs（ P〈0. 01）. Compared with NC-siRNA transfection group,the clone formation ability of si SIRT1 transfection group and the sphere formation ability were decreased,and the tumorigenicity was also significantly decreased（ P〈0. 01）. The expression of SIRT1 in non-CSCs was significantly higher than that in empty vector control group after infection with lentivirus that overexpressed SIRT1,and compared with the control group,the clonal formation ability of non-CSCs,and the tumor formation ability were increased（ P〈0. 01）,and the tumorigenicity was also enhanced in vivo（ P〈0. 01）. Western blotting analysis showed that,after knockdown or inhibition of SIRT1 expression,the expression of SOX2 and Nanog were significantly inhibited（ P〈0. 01）,while the expressions of c-Myc did not change significantly（ P〉0. 05）; Overexpression of SOX2 significantly reversed the effect of knockdown of SIRT1 on clone formation and self-renewal ability,respectively（ P〈0. 01）. Conclusion SIRT1 plays an important role in the self-renewal and tumorigenesis of liver CSCs,which may provide a new target for the treatment of liver cancer.