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Effects of Caveolin-1 siRNA-mediated Silencing of Caveolin-1 on Mouse FBJ-S1 Cells and Studies on GD1a-binding Molecules as Revealed by Immunoprecipitation

Author: WangYiZuo
Tutor: ShanXingDaYe;ShanXingZhenZi
School: Shenyang Pharmaceutical University
Course: Microbial and Biochemical Pharmacy
Keywords: Caveolin Ganglioside GD1a siRNA Hepatocyte growth factor GD1a Immunoprecipitation Mass Spectrometry Two-dimensional electrophoresis
CLC: R73-3
Type: Master's thesis
Year: 2008
Downloads: 14
Quote: 0
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Abstract


The cellar protein -1 (Caveolin-1) is an essential structural component of caveolae, is generally considered to be a tumor suppressor protein. Caveolae mitogenic signals, tumor formation and metastasis have a certain contact. The studies have shown the ganglioside GD1a inhibit murine osteosarcoma cell line FBJ cell transfer, and can induce the expression of Caveolin-1, we used siRNA technology for the study of the role of Caveolin-1 signaling pathway GD1a regulation of cell transfer Caveolin-1 expression in high metastatic FBJ-S1 poor cell-specific inhibition of the expression of Caveolin-1. RT-PCR and western blot results showed that the monoclonal cells stably transfected Caveolin-1 siRNA, siRNA Caveolin-1 gene expression and protein synthesis, the maximum inhibitory effect of up to 80%. Wound healing (the scratches recovery) experimental results show that the reduction of Caveolin-1 expression levels cellular mobile. Under normal culture conditions, the down-regulation of Caveolin-1 expression levels did not affect cell proliferation by WST assay cell survival degrees found. However, under serum-free conditions, with control cells, the cell viability of the caveolin-1 siRNA transfection higher. Using two-dimensional electrophoresis and immunoblotting for the detergent insoluble component (DIF) to be separated and analyzed, the DIF of Caveolin-1 siRNA transfected cells, the phosphorylation of serine protein markedly enhanced the cell signal may be induced. Caveolin-1 expression decreased expression of hepatocyte growth factor significantly increased hepatocyte growth factor is a multifunctional cytokine, can induce cell through the turn and then some other intracellular molecules gene expression level of detection mobile, dispersion and increase the ability of tumor invasion and metastasis. Caveolin-1 expression levels after recovery, import Caveolin-1 cDNA hepatocyte growth factor expression was significantly inhibited in cells transfected Caveolin-1 siRNA. The results of this study show Caveolin-1 has a very important role in the regulation of cell behavior FBJ-S1, and provides further support for GD1a Caveolin-1 inhibits metastasis of this theory. Ganglioside involved in the regulation of a variety of biological phenomena such as cell growth, differentiation, apoptosis and metastasis. The study showed that the ganglioside GD1a by regulation of MMP-9, Caveolin-1, etc. associated with the transfer key molecule to inhibit FBJ cell metastasis. For further elucidate the mechanism of GD1a regulation of tumor metastasis determine GD1a regulation of downstream molecules, the study immunoprecipitation visit GD1a binding molecules. By immunoprecipitation combined with Western blotting, we found Caveolin-1 exist in GD1a antibody precipitated complexes. According to the results of SDS-PAGE, with a molecular weight of approximately 40kD protein specific about the presence precipitated GD1a antibody complexes. GD1a antibody precipitate the complex result of the separation, there are two kinds of low molecular weight (15kD) using two-dimensional electrophoresis, the isoelectric point acidic (about 4) of the protein is obtained from S1 Cell Lysates GD1a antibody precipitated complex higher abundance, higher than LL cells. Has used mass spectrometry analysis identified GD1a antibody precipitated complex of three proteins: actin, positive for vimentin, and Stress-70 protein. Further identified for GD1a antibody precipitated complex ingredients will help to elucidate the molecular mechanisms GD1a regulation of tumor metastasis.

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