Cloning And Characterization Of A Novel Splice Isoform Of Tumor Metastasis Suppressor Gene BRMS1

Breast cancer metastasis suppressor 1, located on chromosome 11q13, is one member of tumor suppressor gene family. BRMS1 was reported to be able to suppress tumor metastasis without affecting primary tumor growth in breast cancer, melanoma and ovarian cancer.However, little knowledge has been known of BRMS1 in hepatocellular carcinoma (HCC). In our previous work, we have found that BRMS1 lost its expression in HCC cells with high metastatic potency. BRMS1 expression is also down-regulated in HCC tissues by comparison with neighboring nontumorous tissues. Overexpression of BRMS1 in HCC cells promoted cell apoptosis, while knockdown of endogenous BRMS1 was able to render cells resistance to apoptosis. All these findings indicated a potential function of BRMS1 in HCC initiation and development.In this study, we reported a novel alternative transcript of BRMS1, BRMS1-Vh. Sequence analysis revealed that BRMS1-Vh lost the whole exon 7 and the translated peptide lost a functional NLS (nuclear localization signal).We first constructed high-molecular-weight and fluorescence-fused proteins to study subcellular localization of BRMS1 and found that the potential of BRMS1-Vh for nuclear import decreased. Further nuclear cytoplasmic fractionation assay demonstrated that more BRMS1-Vh protein was accumulated in cytoplasm rather than nuclear. To analysis the biological function of BRMS1-Vh in HCC cells, we first detected its regulatory effect on NF-κB signaling pathway. By comparison with BRMS1-V1, BRMS1-Vh exhibited a stronger inhibitory role on regulating p65 unclear transport. BRMS1-Vh was also able to interact with HDAC1, HDAC3 and promote the deacetylation of p65. Dual-luciferase report assay further demonstrated that BRMS1-Vh showed a stronger suppressive activity on regulating NF-κB promoter than BRMS1-V1. Over-expression of BRMS1-Vh could reduce the expression levels of endogenous OPN, Bcl-xL, cIAP-2 genes under the control of NF-κB regulation. Transient transfection of BRMS1-Vh could reduce colony formation ratio. Stable expression of BRMS1-Vh showed no effect on cell cycle distribution, but reduced cell growth ratio probably through regulating cell apoptosis. Expression of BRMS1-Vh could sensitize cells to TNFR-induced cell apoptosis. Transplantation of cells stably expressing BRMS1-Vh into nude mice revealed a significant inhibitory effect of BRMS1-Vh in vivo in regulating tumor growth.Taken together, we cloned and characterized a novel transcript of BRMS1. By contrast with BRMS1-V1, BRMS1-Vh tendered to accumulate in cytoplasm, exhibited a stronger inhibitory role in regulating NF-κB signaling. BRMS1-Vh was able to promote cell apoptosis in vitro and inhibited tumor growth in vivo. All these findings will improve our understanding of BRMS1 and provide fundamental evidence for clinical diagnosis, prevention and treatment.