RNA干扰抑制胰岛素样生长因子-1表达并诱导胶质瘤细胞凋亡

Inhibition of insulin-like growth factor 1 expression in glioma cells apoptosis with RNA interference

目的研究RNA干扰效应对胶质瘤C_6细胞株胰岛素样生长因子-1(IGF-1)基因表达的抑制作用及其对C_6细胞凋亡诱导的作用。方法体外化学合成靶向IGF-1基因的小干扰RNA(siRNA),采用脂质体法将siRNA以不同浓度梯度转染C_6细胞株,设非特异的siRNA阳性对照组和未转染siRNA的阴性对照组；同时使用绿色荧光素标记的siRNA异硫氰酸荧光素(FITC)-siRNA转染细胞,于荧光显微镜下观察siRNA转染效率；逆转录聚合酶链反应(RT-PCR)半定量检测siRNA对IGF-1基因表达的抑制作用；流式细胞术检测siRNA诱导细胞凋亡作用。结果荧光显微镜下荧光素标记的siRNA转染组可见到细胞内清晰的绿色荧光,脂质体Oligo- fectamine^(TM)2000的转染效率可＞95%；化学合成的siRNA明显抑制IGF-1 mRNA的表达,各特异性siRNA不同浓度转染组IGF-1 mRNA表达水平可下调约40%～70%,流式细胞术检测转染IGF1-siRNA细胞凋亡率为14．14%,明显高于对照组的0．95%。结论在细胞水平上,化学合成的靶向IGF-1的siRNA可明显抑制IGF-1基因的表达,引致胶质瘤细胞凋亡率明显上升,为进一步利用RNA干扰进行胶质瘤的基因治疗研究奠定基础。

Objective To investigate the inhibitory effect of RNA interference on glioma insulin-like growth factor 1 （IGF-1） gene expression and induction of cell apoptosis. Methods Small interference RNAs （siRNAs） targeting IGF1 gene were synthesized chemically in vitro. C6 cells stably expressing IGF-1 gene were transfected with the siRNA using oligofectamineTM 2000 reagent. Both the nontransfected cells and nonspecific siRNAs transfected cells were taken as negative and positive controls, siRNA transfection efficiency was detected by fluorescent microscopy; down regulation of IGF-1 was demonstrated by RTPCR. Apoptosis of C6 cell was measured by flow cytometry. Results Green fluorescence in the cells was seen clearly in FITC-siRNA transfected group under the fluorescent microscope; the transfection efficiency of oligofectamineTM 2000 was 〉95 %. The expression of IGF-1 mRNA was decreased by 40%-70% in the siRNAs transfected C6 cells with RT-PCR scan analysis as compared with that of the controls. Cell apoptosis could be induced from 0.95% in the control group to 14.14%o at 72 h after transfection. Conclusions At the cellular level, the chemically synthesized siRNAs targeted IGF-1 gene can downregulate the expression of IGF-1, with a remarkable induced apoptosis in comparing with the control cell line. The finding provides a new horizon for the study of glioma gene therapy.