GLUT1基因有效siRNA序列的筛选实验研究

Experiment Study on Screening of Effectual siRNA-mediated GLUT1

目的:设计合成干扰葡萄糖转运蛋白1(glucose transporter 1,GLUT1)基因表达的不同小型干扰RNA(siRNA),筛选出能高效抑制GLUT1基因表达的siRNA。方法:根据人GLUT1 mRNA的序列,设计合成4对不同的针对GLUT1的siRNA,应用脂质体LipofectamineTM2000转染试剂转染293T细胞制作病毒,将4对siRNA转染人舌鳞癌细胞Cal-27,实时荧光定量PCR(Real-time PCR)技术检测GLUT1 mRNA表达;Western印迹法检测GLUT1蛋白表达;18F-FDG检测GLUT1基因干扰后对Cal-27细胞摄取葡萄糖的影响。结果:设计合成的4对siRNA中有2对可有效抑制GLUT1的基因表达以及细胞对葡萄糖的摄取。结论:成功设计合成了针对GLUT1基因的siRNA,并从中筛选出能特异且高效阻断GLUT1表达的siRNA,为进一步应用RNA干扰技术进行GLUT1基因沉默,从而进行肿瘤的基因治疗奠定了实验基础。

Objective： This study aimed to design and synthesize small interfering RNA （siRNA） targeting glucose transporter-1 （GLUT1）, and further screen the siRNA which can specifically and effectively suppresses GLUT1 expression. Methods： Four GLUT1 specific double stranded siRNAs were designed and electroporated into Cal-27 cells by LipofectamineTM 2000 in 293T cells. The transcription level of GLUT1 were analyzed by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）, GLUT1 protein was tested by western blotting and glucose uptake inhibition rates were tested by ^18F-FDG after GLUTI-siRNA. Results： Two of the four customized siRNA manifested effective inhibition on the expression of GLUTI. Conclusion： siRNA targeting GLUT1 can be successfully designed and synthesized, which can specifically and effectively suppresses the expression of GLUT1 gene and glucose uptake. The results may be beneficial to further study on gene therapy of tumors by RNA interfere （RNAi）.