针对AK12的小分子干扰RNA抑制胶质瘤细胞的增殖

Inhibitory Effects of Plasmid-Based siRNA Targeting AKT2 on Tumor Proliferation in C6 Glioma Cells

目的比较靶向性丝／苏氨酸激酶2(AKT2)不同的小分子干扰RNA构建体对脑胶质瘤细胞增殖的抑制作用。方法选择大鼠AKT2的3个siRNA靶序列构建靶向AKr2的siRNA表达质粒,进行对C6细胞的AKT2表达及增殖抑制的研究,蛋白印迹检测AKT2的表达水平,TUNEL法分析细胞凋亡,流式细胞术分析细胞周期变化,~3H-TdR掺入法分析细胞增殖。结果转染siRNA表达质粒组AKT2表达下调72％、88％和91％。各转染组的凋亡率明显增加(x^2：34．218,P<0．001),转染后S期指数较对照细胞明显减少,转染组细胞自第2天起存活率均明显下降(P<0．01),且各个转染组之间存活率也有极显著性差异(P<0．01),以尾部调节结构域转染组最显著。结论靶向AKT2的siRNA表达质粒可以特异性地抑制AKT2的表达,显著抑制肿瘤细胞增殖并诱导细胞凋亡。

Objective To study the inhibitory effects of plasmid-based siRNA targeting AKT2 on tumor proliferation in c6 glioma cells. Methods Three siRNA expression constructs, which target the sequences of AKT2' s 3 different domains of rat (144-164, 728-748 and 1241-1261, respectively), were transfected into C6 cells mediated by Lipofectamine. Western blot was used to detect AKT2 expression. Cell apoptosis was detected for apoptotic index (AI) by using TUNEL method. Cell cycle was analyzed by flow cytometry and cell proliferative activities were measured by ~3H-TdR. Results The expression of AKT2 was decreased by 72%, 88% and 91% respectively in siRNA constructs transfected groups, as indicated by Western blot. Virtually no apoptotic cells were found in parental cells or the cells transfected with empty vector as indicated by TUNEL assay. Apoptosis was increased in siRNA constructs transfected cells. The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in three siRNA constructs transfected cells than those in parental cells and the cells transfected with empty vector. Compared to parental cells and the cells transfected with empty vector, the ~3H-TdR assay indicated that the survival rates of transfected cells dramatically dropped from the second day after implantation. The siRNA transfected cells revealed much lower survival rate than parental and empty-transfected cells. Conclusion The siRNA expressing constructs targeting AKT2 could specifically suppress AKT2 expression, induce gene silencing and inhibit cell growth.