BRMS1过表达促进肝癌SK-HEP-1细胞凋亡

Over-Expression of BRMS1 Promotes Apoptosis in Human Hepatocellular Carcinoma SK-HEP-1 Cells

BRMS1基因和多种肿瘤的转移有关,但其在肝癌发生和转移中的作用及机制仍有待研究.Westernblot的结果显示在SK-HEP-1细胞中检测到差异剪接体2的表达,Hep G2细胞仅检测到差异剪接体1,而L-02和Hep 3B细胞两种差异剪接体都表达,而且在表达差异剪接体1的3株细胞中,Hep 3B细胞差异剪接体1的表达量较高,另两个较低.实时定量PCR对BRMS1基因差异剪接体1的表达进一步检测,发现SK-HEP-1细胞的表达水平最低,Hep 3B最高,L-02和Hep G2介于两者之间.随后以Hep G2细胞的mRNA为模板,扩增人的BRMS1基因差异剪接体1,构建pEGFP-BRMS1质粒,在SK-HEP-1细胞株进行过表达,并用Annexin V-PE和7-AAD双染的流式细胞术研究BRMS1对SK-HEP-1细胞凋亡的影响,结果显示:pEGFP-BRMS1转染组两种染料双阳性,即处于凋亡后期的细胞数目显著高于pEGFP-N2对照组;用PI单染检测存活率,发现pEGFP-BRMS1转染组与pEGFP-N2对照组相比存活率显著降低.上述实验结果表明,BRMS1基因的过表达能够促进SK-HEP-1细胞的凋亡,抑制细胞生存,从而为BRMS1基因抑制肝癌转移机制的阐明提供思路.

BRMS1 has been shown to suppress the metastatic potential of many malignant cell lines,but its mechanisms are poorly understood.Four different cell lines were tested by Western blot for constitutive BRMS1 expression.Only transcript variant 2 was detected in SK-HEP-1 cells,transcript variant 1 in Hep G2,whereas both transcript variants were detected in Hep 3B and L-02;and among those three expressing transcript variant 1,Hep 3B revealed relatively high expression level.In addition,by real-time quantitative PCR analysis of BRMS1 transcript variant 1,the lowest expression was detected in SK-HEP-1 and the highest in Hep 3B.Total RNA was isolated from Hep G2 cells,followed by reverse transcription and PCR amplification,BRMS1 transcript variant 1 was cloned into an expression vector pEGFP-N2,and over-expressed in SK-HEP-1 cells.After incubated with Annexin V-PE in a buffer containing 7-AAD and analyzed by flow cytometry,cells transfected with pEGFP-BRMS1 showed a significant increase in the population of cells Annexin V-PE and 7-AAD positive,indicating that they were in end-stage apoptosis or already dead,over vector-only control.Furthermore,cell death analysis via PI staining also revealed a similar significant difference in cell viability observed between pEGFP-BRMS1 transfectants and control cells.These results collectively indicate that,over-expression of BRMS1 can affect cell viability by promoting cell apoptosis in SK-HEP-1 cells,which may provide novel avenues for future research on the mechanisms of metastasis suppression by BRMS1 in HCC.