c-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro
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Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, invasion, and angiogenesis. The present study was designed to determine the role of c-Met in growth and metastasis of glioma U251 cells using RNA interference (RNAi) technology in vitro. We constructed three kinds of shRNA expression vectors aiming at the c-Met gene, then transfected them into glioma U251 cells by lipofectamineTM 2000. The level of c-Met mRNA was investigated by real-time polymerse chain reaction (RT-PCR). The protein expression of c-Met was observed by immunofluoresence staining and western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. We got three kinds of c-Met specific shRNA expression vectors which could efficiently inhibit the growth and metastasis of U251 cells and the expression of c-Met in U251 cells. RT-PCR, immunofluoresence staining and western blotting showed that inhibition rate for c-Met expression was up to 90%, 79% and 85%, respectively. The expression of c-Met can be inhibited by RNA interference in U251 cells, which can inhibit the growth and metastasis of U251 cell and induce cell apoptosis. These results indicate that RNAi of c-Met can be an effective antiangiogenic strategy for glioma.
Keywordsc-Met Apoptosis Adherence Gene therapy Glioma RNA interference
Hepatocyte growth factor
Hepatocyte growth factor receptor
Roswell park memorial institute
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Methyl thiazole tetrazolium
We thank professor Hong Wang at Henry Ford Hospital for critical reading of the paper. This work was supported by a grant 07JWYQ03 from the Training Excellent Youth Teacher Scientific Research Foundation of University of Shanghai, and a grant 07XYQ01 from the Excellent Youth Teacher Scientific Research Foundation of Shanghai Jiao Tong University of School of Medicine.
- 3.Roccisana J, Reddy V, Vasavada RC, Gonzalez-Pertusa JA, Magnuson MA, Garcia-Ocaña A (2005) Targeted inactivation of hepatocyte growth factor receptor c-Met in (beta)-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of (beta)-cell mass. Diabetes 54:2090–2102. doi: 10.2337/diabetes.54.7.2090 PubMedCrossRefGoogle Scholar
- 5.Camp RL, Rimn EB, Rimm DL (1999) Met expression is associated with poor outcome in patients with axillary lymph node negative breast carcinoma. Cancer 86:2259–2265. doi: 10.1002/(SICI)1097-0142(19991201)86:11<2259::AID-CNCR13>3.0.CO;2-2
- 16.Moriyama T, Kataoka H, Hamasuna R, Yokogami K, Uehara H, Kawano H, Goya T, Tsubouchi H, Koono M, Wakisaka S (1998) Up-regulation of vascular endothelial growth factor induced by hepatocyte growth factor/scatter factor stimulation in human glioma cells. Biochem Biophys Res Commun 249:73–77. doi: 10.1006/bbrc.1998.9078 PubMedCrossRefGoogle Scholar
- 25.Kang CS, Pu PY, Li YH, Zhang ZY, Qiu MZ, Huang Q, Wang GX (2005) An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor. J Neurooncol 74:267–273. doi: 10.1007/s11060-004-8322-z PubMedCrossRefGoogle Scholar
- 26.Christensen JG, Schreck R, Burrows J, Kuruganti P, Chan E, Le P, Chen J, Wang X, Ruslim L, Blake R, Lipson KE, Ramphal J, Do S, Cui JJ, Cherrington JM, Mendel DB (2003) A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res 63:7345–7355PubMedGoogle Scholar
- 27.Bauer TW, Fan F, Liu W, Johnson M, Parikh NU, Parry GC, Callahan J, Mazar AP, Gallick GE, Ellis LM (2005) Insulin like growth factor-I-mediated migration and invasion of human colon carcinoma cells requires activation of c-Met and urokinase plasminogen activator receptor. Ann Surg 241:748–756PubMedCrossRefGoogle Scholar