Liu Hui, Wang Shaochuang, Sun Hangyong, Pan Zeya, Zhou Weiping, Wu Mengchao
The 3rd Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, P.R. China.
Mol Med Rep. 2010 Nov-Dec;3(6):903-7. doi: 10.3892/mmr.2010.355. Epub 2010 Sep 1.
Livin, a member of the inhibitor of apoptosis protein family, is overexpressed in a variety of solid tumors and cancer cell lines. Livin overexpression has been reported in hepatocellular carcinoma (HCC), suggesting the biological significance of livin in HCC progression. However, the mechanisms of livin and the consequence of its down-regulation in HCC have not been fully elucidated. A small interfering RNA (siRNA) eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. It was then transfected into the human HCC cell line SMMC-7721. RT-PCR and Western blotting were used to validate the gene-silencing efficiency of livin in SMMC-7721 cells. Stable clones were obtained by G418 screening. Using multiple cellular and molecular approaches, such as an apoptosis assay, MTT assay, flow cytometry, Western blotting and a migration assay, the effects of livin inhibition on cell growth, migration and the induction of apoptosis in SMMC-7721 cells were observed. The siRNA eukaryotic expression vector specific to livin was constructed by gene recombination, and efficiently decreased the mRNA and protein expression of livin. The targeted inhibition of livin strongly sensitized SMMC-7721 cells to pro-apoptotic stimuli, and was associated with caspase-3 activation. In addition, the MMT assay indicated that the silencing of livin inhibited the cell growth of SMMC-7721 cells by specifically inhibiting cell mitosis. The results also showed that the silencing of livin strongly reduced the invasive capacity of SMMC-7721 cells. The findings suggest that livin expression not only provides HCC cells with increased resistance to apoptotic stimuli, but also contributes significantly to the proliferation and invasive capacity of HCC cells. Inhibition of livin may be a potential targeted approach for the treatment of HCC.
生存素（Livin）是凋亡抑制蛋白家族的成员之一，在多种实体瘤和癌细胞系中过表达。已有报道称其在肝细胞癌（HCC）中过表达，提示生存素在肝癌进展中的生物学意义。然而，生存素在肝癌中的作用机制及其下调的后果尚未完全阐明。通过基因重组构建了针对生存素的小干扰RNA（siRNA）真核表达载体，并对核酸进行了测序。然后将其转染到人肝癌细胞系SMMC - 7721中。采用逆转录聚合酶链反应（RT - PCR）和蛋白质免疫印迹法（Western blotting）验证生存素在SMMC - 7721细胞中的基因沉默效率。通过G418筛选获得稳定克隆。运用多种细胞和分子方法，如凋亡检测、MTT检测、流式细胞术、蛋白质免疫印迹法和迁移检测，观察生存素抑制对SMMC - 7721细胞生长、迁移及凋亡诱导的影响。通过基因重组构建了针对生存素的siRNA真核表达载体，该载体有效降低了生存素的mRNA和蛋白质表达。对生存素的靶向抑制使SMMC - 7721细胞对促凋亡刺激高度敏感，并与半胱天冬酶 - 3（caspase - 3）激活相关。此外，MTT检测表明，生存素沉默通过特异性抑制细胞有丝分裂来抑制SMMC - 7721细胞的生长。结果还显示，生存素沉默显著降低了SMMC - 7721细胞的侵袭能力。这些发现表明，生存素的表达不仅使肝癌细胞对凋亡刺激的抵抗力增强，而且对肝癌细胞的增殖和侵袭能力也有显著贡献。抑制生存素可能是治疗肝癌的一种潜在靶向方法。
