Primary Study The Effect Of HHBrk1 On Lung Carcinoma Cell Biological Behaviors And Its Interaction With WAVE1

Human homology of Brick1(hHBrk1) was primarily identified to be an over-expressed novel gene in theαparticles-transformed human bronchial epithelial cells in our laboratory, owing to its high homology with maize Brick1 gene, so named as hHBrk1. hHBrk1 was located in human chromosome 3p25.3-24.1 regions, composed of three exons, two introns and 75 amino acid of coding protein which was about 9kD, this coding protein was also name HSPC300, C3ORF10 or MDS027. hHBRK1 is highly conserved through out the mammalian and plant kingdom. Computer assisted analysis of hHBrk1 47-75 amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential forα-helical coiled coil formation. The coiled coil formation is important domain of leading to protein interaction.Our previous results showed that hHBrk1 gene expressed ubiquitously in normal human tissues derived from adult and fetus, and more expressed in lung cancer than adjacent cancer sider . hHBrk1 protein distributed diffusely in cytoplasm of 95D lung cancer cell, and became enrichments at the tips of protruding lamellipodia. As well as were overlapping with F-action at the leading edge of living cells. By using GST pull-down assay and mass spectrum methods, myosin VI was found to be helpmate molecule of hHBrk1. hHBrk1 could interact with myosin VI which play an important function on the migration of tumor cell.All of these indicated that hHBrk1 is an actin-associated new gene. It could play an important role on tumor permeable transfer spread process.The current study found that hHBrk1/HSPC300 was one number of WAVE/Scar heterotetrameric complexity. Participate in regulating nucleation of polymerization, cell movement as well as play a role in cargo transport all of these basic being phenomena via keep stability of WAVE heterotetrameric complex.WAVE protein family, exists in a state of heterotetrameric complex,is a key protein to regulating actin assembly.When environment changed, activated Rac1 which interact with Nck binding protein could lead to disassembling the trans-inhibited WAVE1 complex. The WAVE1 protein, associated with hHBrk1, was released from the complex. Activated Scar/WAVE1 protein binds and induces a conformational change in the Arp2/3 complex, also this key component could regulating nucleation of polymerization and leading to actin assembly.But the reciprocity spot of hHBrk1 and WAVE have not clear. This manuscript will report our primary study the effect of hHBrk1 on lung carcinoma cell biological behaviors and its interaction with F-actin associated protein----WAVE ( WASP-family Verprolin homology protein). and acquired result was as follows.1.Effect of silencing hHBrk1on lung carcinoma cell biological behaviors1) In order to further investigate the function of hHBrk1 on tumorogenesis and cell malignant transformation, RNA interference technique was used to silence the expression of hHBrk1. Stable transfectants were selected from the 95D cells transfected with hHBrk1 siRNA-expressing vector. The expression of hHBrk1 was significantly suppressed by siRNA.2) The over-expressed hHBrk1 gene in 95D cell was silenced by RNAi technique. No differences were found in proliferation and clone formation between hHBrk1 silencing cells and control cells. But the ability of migration in hHBrk1 silencing cells was impaired. The results indicate the hHBrk1 may not be involved in tumor cell proliferation and differentiation .But it relate to movement of cells, and associate with migration of the lung cancer cells.3) The morphogenesis change was also observed in 95D cells with hHBrk1 silencing. Silencing hHBrk1 gene resulted in 95D cells bigger and the configuration was abnormity. The cell membrane was thinner, the extended pseudopodia were decreased, and the length of pseudopodia became lack. The reduced F-actin enrichments was found at the leading edge of silencing hHBrk1 gene living cells compared with control cells with confocal microscope analysis. But no affect on the expression and location of Golgi, Lysosome and Endoplasmic. The morphogenesis change after silencing hHBrk1 gene was consistent with F-actin enrichments reduced at the leading edge of living cells.4) MAPK signal transduction passway of varing proteins was analyzed by western blot, when hHBrk1 gene was in Silencing state, we found that phosphorylated Erk1/2 expression was decreased, while the phosphorylated JNK and p38 expression was increased. This result was consistent with the ability of migration impaired in hHBrk1 silencing cells. It demonstrated that hHBrk1 protein could improve the ability of migration of carcinoma cell via Erk1/2 signal transduction pathway.At same time, increased the ability of stress response by JNK and p38 signal transduction pathway.2. Identify the interaction of hHBrk1 with WAVE: Interaction of hHBrk1 with WAVE1 was detected by co-immunoprecipitation . In order to explore the reciprocity spot, mutant WAVE1 vectors were constructed and co-immunoprecipitation results indicated the C-terminal region of hHBrk1 GST fusion protein may bind with the SHD( Scar homology domain) domain of WAVE1. The C-terminal region of hHBrk1 comprised a heptad repeat (HR) motif with a potential forα-helical coiled coil formation.This manuscript reported hHBrk1 may involve in migration of the 95D cells, and via MAPK signal transduction pathway. Additionally,hHBrk1 may interact with WAVE1, and interaction may via binding of the heptad repeat (HR) motif of hHBrk1 and the SHD( Scar homology domain) domain of WAVE1. Results of this program provided with convincingly experimental evidences for further exploring mechanism of hHBRK1 influencing on tumor permeable transfer spread and become anticancer target.