RNA干扰IDH2基因对HCT-8细胞增殖能力的影响

Influence of IDH2 on proliferation of colon cancer cells HCT-8 by siRNA

目的:研究RNA干扰(RNAi)对结肠癌HCT-8细胞IDH2基因表达的抑制作用及干扰后对HCT-8细胞增殖的影响,为结肠癌的基因靶向治疗提供理论依据。方法:实验分siRNA干扰质粒转染组、阴性质粒对照组和HCT-8细胞空白对照组。构建靶向基因IDH2的siRNA真核绿色荧光载体重组质粒PGPU6/GFP/Neo-IDH2,利用FuGENE HD转染剂转染HCT-8细胞,通过荧光定量PCR检测IDH2mRNA的表达情况及MTT法检测结肠癌HCT-8细胞增殖变化。结果:在荧光显微镜下进行细胞计数,细胞转染率为74%。实时荧光定量PCR检测,siRNA重组质粒PGPU6/GFP/Neo-IDH2对IDH2基因表达的抑制率为85.02%,与空白对照组比较差异有统计学意义(P<0.05)。MTT检测,干扰质粒转染组细胞的增殖能力明显低于空白对照组(P<0.05)。结论:抑制IDH2基因的表达可影响结肠癌细胞的增殖能力,提示IDH2基因在结肠癌的发生发展中起重要作用,抑制IDH2基因的表达可能成为一种治疗结肠癌的方法。

Objective To analyze the inhibitory effect of RNA interference （RNAi） on the expression of IDH2 gene of colon cancer cells HC7-8 and the influnce in the proliferation of colon cancer cells in vitro, and to provide a theoretical basis for colon cancer targeted therapy. Methods The experiment was divided into siRNA plasmid transfection, negative plasmid control and HCT-8 cells control groups. The recombinant plasmid which contained the siRNA of the IDH2 gene in the green fluorescence eukaryotic vector （PGPU6/GFP/Neo-IDH2） and the negative control were constructed and then they were transfected into colon cancer cells by using FuGENE HD Transfection Reagent. The expression of IDH2 gene mRNA was detected by real-time PCR. The proliferation abilities of colon cancer cells were detected by MTT. Results Counted by fluorescence microscope, the transfection rate of cells was 740//oo. The inhibitory rate of IDH2 mRNA in PGPU6/GFP/Neo-IDH2 group was 85.02~, which hadsignificant difference compared with HCT-8 cells control group （P^0.05）. Compared with neagtive control group, the proliferation ability of colon cancer cells was deceased by MTT assay （P^0.05）. Oonelusion Inhibition of the expression of IDH2 gene can infect the proliferation ability of colon cancer cells. It shows that IDH2 gene plays a role in the development of colon cancer and it may be beneficial in finding a new therapy for colon cancer.