SIRT2对裸鼠原位肝癌生长和肺转移的影响及其机制研究

Effects of SIRT2 on Growth and Lung Metastasis of Orthotopic Liver Cancer in Nude Mice and Its Mechanism

该文旨在探讨沉默信息调节因子2(silent information regulator 2,SIRT2)基因敲低对裸鼠原位肝癌生长和肺部转移的影响。应用杀稻瘟菌素筛选构建稳定敲低SIRT2基因的SK-Hep-1细胞,应用定量逆转录PCR(q RT-PCR)和Western blot检测其敲低效率。将稳定敲SIRT2基因的细胞(SIRT2-sh RNA-SK-Hep-1)和对照组细胞(sh Cont-SK-Hep-1)原位注入裸鼠肝脏,8周后处死裸鼠并取出肝脏和肺组织,比较两组裸鼠肝癌肿瘤的大小及肺转移结节的数量。应用Western blot和免疫组织化学检测两组裸鼠肝癌组织中SIRT2、p-AKT、AKT、p-GSK3β、GSK3β、activeβ-catenin和β-catenin的蛋白质水平。结果显示,稳定敲低SIRT2基因的SK-Hep-1细胞系构建成功。裸鼠原位肝癌肺转移模型中,相比sh Cont-SK-Hep-1组,SIRT2-sh RNA-SK-Hep-1组裸鼠原位肝癌体积减小(P<0.05),肺转移结节数量明显减少(P<0.05)。Western blot结果表明,敲低SIRT2基因的裸鼠组p-AKT、p-GSK3β、activeβ-catenin、β-catenin蛋白质水平降低,GSK3β水平升高,AKT水平无差异;免疫组织化学结果表明,敲低SIRT2基因的裸鼠组p-AKT、p-GSK3β、β-catenin蛋白质水平降低。本研究结果提示,在裸鼠原位肝癌肺转移模型中,敲低SIRT2基因可以通过激活AKT的表达影响GSK3β/β-catenin信号通路,从而抑制人肝癌细胞的生长和肺转移。

The aim of this study was to investigate the effects of SIRT2 on growth and lung metastasis of orthotopic liver cancer in nude mice. SK-Hep-1 cells with stable knockdown of SIRT2 were screened with blasticidin and its knockdown efficiency was detected by qRT-PCR and Western blot. SIRT2-shRNA-SK-Hep-1 and shCont-SK-Hep-1 cells were respectively injected into the orthotopic liver of nude mice. The liver and lung tissues of nude mice were removed after 8 weeks of injection. The size of orthotopic liver cancer and the number of lung metastatic nodules were compared between the two groups. Next, the protein levels of SIRT2, p-AKT, AKT, p-GSK3β, GSK3β, active β-catenin and β-catenin in nude mice were detected by Western blot and immunohistochemistry. The results showed SK-Hep-1 cell lines with stable knockdown of SIRT2 were successfully established. Compared with shCont-SK-Hep-1 group, the volume of liver cancer in SIRT2-shRNA-SK-Hep-1 group was decreased （P〈0.05） and the number of lung metastatic nodules in SIRT2-shRNA-SK-Hep-1 group was decreased significantly （P〈0.05）. Western blot showed that the protein levels ofp-AKT, p-GSK3β, active β-catenin, β-catenin in the SIRT2 knockdown group were decreased, the protein level of GSK3β was increased, the protein level of AKT was no difference. Immunohistochemistry showed that the protein levels of p-AKT, p-GSK3β, β-catenin in the SIRT2 knockdown group was decreased. The results indicated that SIRT2 knockdown can inhibit the growth and lung metastasis of human hepatocarcinoma cells by activating AKT, subsequently influencing on GSK3β/β-catenin signaling pathway in orthotopic liver cancer of nude mice.