Inhibition Of C-met Gene Expression And Induction Of Apoptosis By C-met SiRNA In HepG2 Cells

Primary liver cancer remains one of most common cancers in China. Primary liver cancer has relative poor prognosis due to high relapse and metastasis rate . C-met is involved in regulation of cell growth and apoptosis in many tumor cells and can be one of molecular targets in targeted therapy of cancer treatment. It has been shown that c-met plays an important role in the development of primary liver cancer. However, studies on c-met targeted therapy are not common.The development of cancer is a process of complex and consecutive changes. The key is changes in genes during the process of normal cells converted to cancer cells. Cell growth is modulated by many signals in normal condition while uncontrolled cell growth is the consequence of imbalanced regulation. Expression of oncogenes can promote cell growth . Oncogenes can cause hyperplasia or carcinogenesis while they undergo changes in structure or overexpression and subsequently illustrate strong action in growth promotion.C-met gene is located in 7q31 and is larger than 110kb including 21 exons. The coding protein of c-met is receptor of hepatocyte grow factor. Mature protein is located on cell membrane. α subunit of 50kDa and β subunit of 145kDa are linked together by disulfide bond to form a dimer of 190kDa. C-met receptor developed autophosphorylation and activation of its tyrosine kinase after HGF binding and subsequently phosphorylation of substrate protein. C-met expression is markedly elevated in liver cancer compared to normal liver tissue and is related to differentiation , metastasis and survivaltime. High expression of c-met is related with higher metastasis rate and shorter survival time in liver cancer patients.RNAi is a phenomenon of genetic interference mediated by double strand RNA which can degrade mRNA specifically and effectively and cause post-transcriptional gene silencing. Recent research showed 21-25 siRNA (small interference RNA) can mediate specific gene silencing in mammalian cells. RNAi is high specifically and effectively and can be new technology in gene function study and gene therapy. The maximal inhibition concentration is only ten or more nmol. Now siRNA has been a strong tool in knockdown of specific gene in mammalian cells. At present antisense oligonucleotide has low efficiency and reproducibility as a common method inhibiting specific gene expression. RNAi has showed promising future in gene therapy.In our study we transfected c-met siRNA into HepG2 cells and observed inhibition of c-met gene expression by c-met siRNA. We also studied whether c-met siRNA can induce apoptosis of HepG2 cells and whether combination of gemcitabine , docitaxel or Aidi Zhusheye can enhance its inhibition on growth of HepG2 cells.In the paper the study was separated into three parts.Part one: Inhibition of c-met gene expression by c-met siRNA in HepG2 cellsAim: Synthetic c-met siRNA was transfected into HepG2 cells to observe inhibition of c-met mRNA and protein.Methods: Fluorescence quantitative PCR and immunohistochemistry.Results: SiRNA A transfection in HepG2 cells after 48 hours didn' t inhibit c-met mRNA while siRNA B inhibit c-met mRNA markedly. Inhibition rate of c-met mRNA is proportioned to siRNA B concentration and is strongest from 48 to 72 hours after transfection. C-met protein is expressed on the membrane or in the plasma of HepG2 cells. SiRNA B transfection can inhibit c-met proteinexpression by immunohistochemistry.Conclusion:C-met is high expressed in HepG2 cells. SiRNA B can inhibit c-raet mRNA and protein expression and is proportioned to concentration of SiRNA B.Part two: Induction of apoptosis by c-met siRNA in HepG2 cellsAim: Synthetic siRNA was transfected into HepG2 cells to observe apoptosis.Methods: Microscopic observation , DAPI staining, annexin V/PI flow cytometry and MTT assay.Results: Microscopic observation and DAPI staining showed siRNA B can induce HepG2 cell apoptosis . Annexin V/PI flow cytometry and MTT assay showed apoptosis is proportioned to concentration of siRNA B.Conclusion: siRNA B can induce HepG2 cell apoptosis and apoptosis is proportioned to concentration of siRNA B.Part three: the role of c-met siRNA in the cell growth inhibition of HepG2 cells combined with different drugs.Aim: C-met siRNA was transfected into HepG2 cells combined with gemcitabine , docitaxel and Aidi Zhusheye to observe growth inhibition of HepG2 cells.Methods:MTT assays.Results: Growth inhibition of HepG2 cells is proportioned to concentration of gemcitabine and combination of siRNA B can enhance growth inhibition of gemcitabine. Growth inhibition of HepG2 cells is proportioned to concentration of docitaxel and combination of siRNA B can enhance growth inhibition of docitaxel. Growth inhibition of HepG2 cells is proportioned to concentration of Aidi Zhusheye and combination of siRNA B can enhance growth inhibition of Aidi Zhusheye.Conclusion: Combination of siRNA can enhance growth inhibition of gemcitabine in HepG2 cells. Combination of siRNA can enhance growth inhibitionof docitaxel in HepG2 cells. Combination of siRNA can enhance growth inhibition of Aidi Zhusheye in HepG2 cells.Conclusion:1. c-met is high expressed in HepG2 cells.2. SiRNA B can inhibit c-met mRNA expression and is proportioned to concentration of SiRNA B.3. SiRNA B can inhibit c-met protein expression and is proportioned to concentration of SiRNA B.4. siRNA B can induce HepG2 cell apoptosis and apoptosis is proportioned to concentration of siRNA B.5. Combination of siRNA can enhance growth inhibition of gemcitabine , docitaxel and Aidi Zhusheye in HepG2 cells.