Role Of The Novel Gene BNIPL Involved In The Growth And Metastasis Of Human Hepatocellular Carcinoma Cells

BNIPL is the novel gene isolated by our laboratory that could inhibit the proliferation of the cancer cells. It has two variants, BNIPL-1 and BNIPL-2. The difference is that BNIPL-1 is shorter by 82 amino acids at N-terminus than that of BNIPL-2. Both BNIPL-1 and BNIPL-2 contain a BCH domain (BNIP2-Cdc42GAP-Homology) and also an arginine-patch motif conserved in Rho family and related proteins.BNIPL-1 could inhibit the cell growth via interacting with two cell proliferation-related proteins MIF and GFER. To explore the roles of BNIPL-1 in cell growth, we established Hep3B-Tet-on-BNIPL-1 stable cell line in which expression of BNIPL-1 could be induced by doxycycline. The differential expression profiles of 588 known genes were determined using Atlas human cDNA expression array. Fifteen genes were differentially expressed following the overexpression of BNIPL-1. Most of them were involved in cell proliferation or cell apoptosis. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Cell proliferation activity (MTS) assay showed that the overexpression of BNIPL-1 could suppress Hep3B cell growth in vitro. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway(s).According to the previous results, BNIPL-2 could interact with Cdc42GAP and suppress the GAP activity of Cdc42GAP toward Cdc42. BNIPL-2 also had a BCH domain (BNIP-2 and Cdc42GAP homology), which could induce short and long pseudopodia, and subsequently was needed to trigger cell migration. So we presume BNIPL-2 could be involved in cell migration and cancer metastasis. On the one hand, to understand the implications of BNIPL-2 in the development of human hepatocellular carcinoma (HCC), we investigated the downstream signaling events regulated by BNIPL-2 using Atalas human cDNA expression arrays in Hep3B-Tet-on-BNIPL-2 cells. Among 588 known genes spotted on the Atlas membrane, fifteen genes showed altered expression levels following overexpression of BNIPL-2. These genes were involved in cell cycle, cellapoptosis, signal transduction and transcription. The data indicates that BNIPL-2 may play its part roles in cell growth. On the other hand, in order to investigate the effects of BNIPL-2 in cell migration and cancer metastasis, a full-length human BNIPL-2 cDNA was transfected into the metastatic human hepatocellular carcinoma cells with low metastatic potential (MHCC97-L). The effect of BNIPL-2 on cell invasion and metastasis in vitro and in vivo and the molecular mechanisms were examined. In vitro analysis showed BNIPL-2 could increase cell invasion and promote cell migration. In addition, the overexpression of BNIPL-2 could enhance the rate of intrahepatic and pulmonary metastasis in nude mice. BNIPL-2 could also maintain the high level of Cdc42-GTP, the active Cdc42 form, and then altered cellular morphology. Western blotting and immunocytochemistry assay demonstrated that there was the significant upregulation of MMP-2 and CD44 in BNIPL-2 transfectants. So overexpression of BNIPL-2 could promote the metastasis of HCC through regulating cell migration, invasion and adhesion.In conclusion, the overexpression of BNIPL-1 could suppress Hep3B cell growth in vitro, and we could presume the signal transduction pathways regulated by BNIPL-1 in cell proliferation and apoptosis. But BNIPL-2 could not suppress Hep3B cell growth significantly. The metastasis analysis in vitro and in vivo showed that overexpression of BNIPL-2 could promote the metastasis of HCC through regulating cell migration, invasion and adhesion. The different role of BNIPL-1 and BNIPL-2 in HCC may result from the fact that BNIPL-1 is shorter by 82 amino acids at N-terminus than BNIPL-2. These findings provide a clue to investigate the further mechanisms of BNIPL in cell growth and cancer metastasis.