Expression Of Brms1 Gene In Hepatocellular Carcinoma And Apoptotic Activity Of

Tumors metastasis is a major cause of death in Hepatocellular Carcinoma(HCC) patients and at the same time a complex multistep process,during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs.A better understanding of the molecular mechanisms associated with metastasis should lead to improvements in therapeutic strategies for HCC.BRMS1,one of the newly discovered metastasis suppressor genes,has been shown to suppress the metastatic potential of many malignant cell lines,but its mechanisms are poorly understood.Previous studies indicate that BRMS1 may function as a transcriptional corepressor of antiapoptotic genes regulated by NF-κB via interaction with mSin3·HDAC complex,and that BRMS1 can inhibit metastasis by decreasing both the number of cells that initially survive in a metastatic site as well as inhibiting outgrowth of metastases in these sites.Here,we sought to explore whether BRMS1 is a common key gene as metastasis-suppressor in HCC,and also inhibits metastasis of HCC via negative regulation of cell survival after the loss of cellular adhesion.Based on these hypotheses,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.Detailed understanding of the relationship between apoptosis and metastasis suppression in HCC,through the study of BRMS1,may provide novel avenues for future metastasis research.