BACKGROUND:Human gliomas are more likely to express basic fibroblast growth factor-2(FGF-2), insulin-like growth factor-1(IGF-1),and IGF-1 receptor(IGF-1R) than normal brain tissue.These factors activate signal transduction systems of Ras/MAPK and PI3K/Akt,which promote glioma growth. OBJECTIVE:To utilize RNA interference(RNAi) technique to down-regulate FGF-2,IGF-1,and IGF-1R gene expression,and to investigate the effects of these genes on rat C6 glioma cells,as well as the feasibility of RNAi for treating glioma. DESIGN,TIME AND SETTING:This neurooncological,randomized,controlled,in vivo and in vitro experiment,which used RNAi methodology,was performed at the Laboratory of Molecular Biology, Institute of Biochemistry,Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS:Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences.Small interfering RNA(siRNA) was synthesized by Shanghai GenePharma.Anti-IGF-1,anti-IGF-1R,anti-FGF-2,anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology,USA.Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center,Chinese Academy of Sciences. METHODS:C6 glioma cells were transfected with siRNA,which was chemically synthesized in vitro to correspond to endogenous FGF-2,IGF-1,and IGF-1R genes.The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR,and protein expression was determined by Western blot analysis.C6 glioma cell proliferation was observed using a growth curve. C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry.C6 glioma cell growth regression was observed by transwell migration assay.In addition,nude mouse subcutaneous tumor models were used in this study.For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA,two blank control groups,with six mice each,were set up:A(2.5μg siRNA was injected one week after C6 cells were inoculated,i.e.,when tumor volume reached 8 mm×8 mm) and B(siRNA was injected at the same time with C6 cells were inoculated.To study the effects of FGF-2 siRNA, the groups consisted of a blank control group,negative control group,2.6μg siRNA group,4μg siRNA group,and 5.3μg siRNA group,with six mice each. MAIN OUTCOME MEASURES:mRNA and protein inhibition ratio of FGF-2,IGF-1,and IGF-1R;C6 glioma cell proliferation,apoptosis,and cycle growth arrest;C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS:All siRNA constructs proved to be effective.After 48 hours,transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio>80%and an IGF-1 gene inhibition ratio of approximately 70%.Protein expression levels for FGF-2,IGF-1,and IGF-1R decreased in a dose-dependent manner following siRNA transfection,with an inhibition rate>85%,60%,and 50%, respectively.C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA.The apoptosis rate of C6 glioma cells induced by FGF-2,IGF-1,and IGF-1R siRNA was 39.96%,15.07%, and 22.47%,respectively(P<0.01).Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration.At 30 days after intratumoral injection of 2.6,4,and 5.3μg FGF-2 siRNA,tumor growth regression rate of FGF-2 siRNA was 56%,67%,and 86%,respectively. The tumor growth regression rate was 71.88%and 45.71%,respectively,when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation.When IGF-1 or IGF-1R siRNA was intratumorally injected during C6 glioma cell transplantation,the tumor growth regression rate was 78.13%and 74.29%,respectively. CONCLUSION:siRNA transfection downregulated gene expression of FGF-2,IGF-1,and IGF-1R. In addition,siRNA treatment markedly suppressed glioma cell proliferation,growth,and migration, and concomitantly reduced subcutaneous tumorigenicity. BACKGROUND: Human gliomas are more likely to express basic fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), and IGF- Factors activate signal transduction systems of Ras / MAPK and PI3K / Akt, which promote glioma growth. OBJECTIVE: To utilize RNA interference (RNAi) technique to down-regulate FGF- 2, IGF- 1, and IGF- 1 R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING: This neurooncological, randomized, controlled, in vivo and in vitro experiments, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS: Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA (siRNA) was synthesized by Shanghai GenePharma. Anti-IG F-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six weeks old BALB / c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS: C6 glioma cells were transfected with siRNA, which was benzyl synthesized in vitro to correspond to endogenous FGF-2, IGF-I, and IGF-1 R genes.The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve. C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up: A (2.5 μg siRNA was injected one week after C6 cellswere inoculated, ie, when tumor volume reached 8 mm × 8 mm) and B (siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, Negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group with six mice each. MAIN OUTCOME MEASURES: mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1R; C6 glioma cell proliferation: apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS: All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol / L siRNA resulted in a FGF-2 or IGF- gene inhibition ratio> 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose- dependent manner following siRNA transfection, with an inhibition rate > 85%, 60%, and 50%, respectively.C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA.T The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1 and IGF-1R siRNA was 39.96%, 15.07%, and 22.47%, respectively (P <0.01) .Transfection of 200 nmol / 1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4 and 5.3 μg FGF-2 siRNA, tumor growth rate of FGF-2 siRNA was 56%, 67%, and 86% respectively. IGF-1 or IGF-1R siRNA was intratumorally injected during 1 week after C6 glioma cell transplantation CONCLUSION: siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R. In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.