生长因子受体反义基因逆转肝癌细胞的恶性表型

The antisense gene of growth factor receptor reversing the malignant phenotype of human hepatoma cells

目的应用反义技术原理将生长因子受体反义基因,导入人肝癌SMMC-7721细胞,阻断肝癌细胞生长因子与其受体的相互作用,研究其对肝癌细胞恶性表型的逆转作用。方法构建人胰岛素样生长因子Ⅱ型受体(IGF-ⅡR)正、反义基因重组真核表达质粒,用磷酸钙-DNA共沉淀方法转染人肝癌细胞株SMMC-7721,经G418培养基筛选得到稳定的抗性细胞。采用Northern杂交检测反义基因转染细胞内源性IGF-ⅡR基因mRNA转录的抑制,同时观察转染细胞克隆形成率,细胞生长曲线和软琼脂生长能力的变化。结果 IGF-ⅡR反义基因转染后,肝癌细胞的内源性IGF-ⅡR基因mRNA转录受到有效抑制;各组转染细胞克隆形成率分别为:空载体组22.0±3.6,正义组13.3±2.1,反义组4.3±2.1,反义基因转染细胞克隆形成率明显降低;软琼脂培养基细胞集落形成数为:SMMC-7721组254.8±15.9,空载体组196.4±30.4。正义组99.4±23.0,反义组0.0±0.0,说明反义基因转染细胞失去在软琼脂上生长的能力;但细胞生长曲线无明显变化。结论应用反义策略干扰肝癌细胞生长因子受体基因的异常高表达状态,可以有效阻断配体与受体间作用的生长刺激信号环路,一定程度地抑制肝癌细胞的恶性表型。

AIM According to the antisense theory, we transferred the growth factor receptor antisense gene into human hepatoma cell line SMMC-7721 to block the interaction between ligand and its receptor, to observe the reversing effects of its malignant phenotype. METHODS The recombinant eukaryotic expression plasmids of IGF-Ⅱ R sense and antisense gene were transferred into SMMC-7721 human hepatoma cell line using calcuim phosphate coprecipitation, which were selected in DMEM supplemented with 500mg/L G418 (Geneticin) for 2-3 weeks. Then the total number of colonies formed was counted. The effect of IGF-Ⅱ R antisense gene transfection on endogenous IGF-Ⅱ R mRNA levels was examined by Northern blot, and the growth rate and the ability of the transfected cells to form colonies in soft agar medium were also examined. RESULTS The hepatoma cells transfected with IGF-Ⅱ R antisense gene exhibited significant reduction in endogenous IGF-Ⅱ R messenger RNA (mRNA) levels. The numbers of geneticin-resistant colonies formed after transfection with pCMVneo vector, sense and antisense constructs were 22.0±3.6, 13.3±2.1 and 4.3±2.1 respectively. The transfection of IGF-ⅡR antisense cDNA suppressed the formation of colonies of SMMC-7721 cells resistant to G-418. The numbers of colonies formed in soft agar by parental and transfected cells (pCMVneo, sense and antisense) were 254.8±15.9, 196.4±30.4, 99.4±23.0 and 0.0±0.0 respectively. The antisense construct transfected cells lost their ability for anchorageindependent growth in soft agar. No significant difference in the growth rate of the various populations of transfected cells was observed compared with the parental cells. CONCLUSION To interfere with the growth factor receptor gene expression of hepatoma cell by antisene strategy can effectively block ligand to receptor signal transduction pathway and reverse certain aspects of the cell malignant phenotype.