Cyclin E RNA干扰载体的构建及其对EC9706细胞Cyclin E表达的影响

Construction of RNAi vector of Cyclin E and its effection on Cyclin E expression of EC9706 cell

目的:构建CyclinE基因干扰载体,转染食管癌EC9706细胞,观察其对CyclinE基因表达的影响。方法:设计、合成靶向CyclinE基因siRNA,退火成双链后与pRNAT-CMV3.1/Neo载体连接。转染EC9706细胞后,倒置荧光显微镜下观察细胞转染效果,RT-PCR、Western-blot分别检测CyclinE基因mRNA和蛋白表达水平。结果:构建的2个CyclinE基因siRNA载体插入序列与所设计序列均一致;转染EC9706细胞后,干扰1组、干扰2组与无关siRNA对照组均产生绿色荧光,空白对照组无荧光产生。干扰1组、干扰2组与空白对照组和无关siRNA对照组CyclinEmRNA相对表达量比较,差异有统计学意义(F=15.381,P=0.002),且干扰1组、干扰2组与空白对照组和无关siRNA对照组比较,CyclinEmRNA相对表达量均降低(P均<0.05)。结论:构建的靶向CyclinE基因RNA干扰载体能有效抑制转染细胞CyclinE基因的表达。

Aim： To construct the RNA interference vector of Cyclin E and transfect into EC9706 cell lines,and observe the supression effects on the expression of Cyclin E in transfected cell. Methods： Two pairs of small interfering RNA targe-ting to Cyclin E were designed. The annealed products （ ds1,ds2） were ligated into RNA interference vector. The recombi-nant vectors were transfected into EC9706. GFP expression of transfected cells was detected by fluorescent microscopy. mR-NA and protein expression of Cyclin E were measured by RT-PCR and Western-blot assay. Results： The 2 inserted se-quences which combined into siRNA vector were the same with the target sequences designed previously. Green fluorescence of the transfected cells could be observed under fluorescent microscopy in interfered group 1,interfered group 2 and non-siR-NA associated group but no green fluorescence in blank control group. There were significant differences in the expression of Cyclin E mRNA among the four groups（ F = 15. 381,P = 0. 002） . And the expression of mRNA in the interfered group 1 and 2 were significantly decreased compared with those in non-siRNA associated group and blank control group （ P 0. 05） . Con-clusion： The recombinant vector could effectively suppress the expression of Cyclin E gene in RNA level.