RNA干扰抑制多形性胶质母细胞瘤核因子-κB的表达作用及其机制

Inhibitory effect of RNA interference on the expression of nuclear factor - κB in glioblastoma multi-forme and its mechanism

目的探讨RNA干扰（RNAi）抑制多形性胶质母细胞瘤核因子-κB（NF-κB）的表达作用及其机制。方法构建慢病毒p65特异性靶序列及其表达载体，并将其包装至293T细胞，之后转染A172细胞，小鼠随机分为空白组、模型组、低和高RNAi组及阴性对照组。在小鼠皮下造模14d后低和高RNAi组小鼠经尾静脉分别注射RNAi慢病毒载体颗粒50、100μl（1×10^5TU／μl）、阴性对照组注射空白载体病毒颗粒100μl（1×10^5TU／μl）。结果慢病毒转染A172细胞后可以显著抑制其增殖（P=0．000），与对照组比较，高剂量组存活率为60％。NF-KBp65特异性靶序列定向干扰A172细胞，3个RNAi组中p65蛋白的表达水平显著下降（P=0．000）。动物实验结果显示，除空白组外，其余所有组小鼠体重自植瘤9d后体重开始出现下降，高低剂量慢病毒干扰组小鼠体重在第18天体重出现回升，其中体重回升高剂量干扰效果略优于低剂量干扰组。模型组小鼠肿瘤体积显著高于两个RNAi组（P=0．000），阴性慢病毒对照组小鼠肿瘤体积无显著缩小。模型组和阴性对照组肿瘤组织中p65处于高表达水平，RNA高低剂量干扰组p65表达水平显著下降（P=0．000）。炎性因子白细胞介素（IL）-1β和肿瘤坏死因子-α（TNF-α）在模型组中处于高表达水平，使用p65干扰RNA后能够有效抑制IL-1β和TNF-α的表达（P=0．000）。结论特异性干扰NF．KBp65蛋白表认后能够有效改善小鼠磨下种棺膣质瘤A172带来的荷瘤压力和隆低小鼠机体赍忡反府．

Objective To investigate the inhibitory effect of RNA interference on the expression of nuclear factor - κB （ NF - κB） in glioblastoma muhiforme and its mechanism. Methods The specific tar- get sequence and expression vector of lentivirus p65 were constructed and packed into 293T cells and then transfected into A172 cells. The mice were randomly divided into blank group, model group, low and high RNAi group and negative control group. After 14 days of subcutaneous model of mice, the RNAi lentivirus carrier particles were injected into the tail vein of the low and high RNAi mice, respectively, 50 and 100 pJ （ 1 ×10^5 TU/μl） , and the negative control group was injected with the blank carrier virus panicles 100 μl （ 1×10^5 TU/μl）. Results Lentivirus transfected A172 cells significantly inhibited the proliferation （P = 0. 000）. Compared with the control group, the survival rate of the high dose group was 60%. The specific target sequence of NF - KB p65 interfered with A172 cells, and the expression level of p65 protein in the 3 RNAi groups decreased significantly （ P = 0. 000 ）. The results of animal experiments showed that the weight of all the mice in the other groups began to decline after 9 d, and the weight of the mice in the low dose lentivirus interference group increased in Eighteenth days, and the effect of high dose weight recovery was slightly better than the low dose interference group. The tumor volume of the mice in the model group was significantly higher than that of the two RNA interference group （ P = 0. 000） , and the tumor volume of the negative lentivirus control group was not significantly reduced. The detection of NF - KB 1365 protein level in tumor tissue showed that p65 was high in model group and negative control group, and the level of p65 expression in RNA high and low dose interference group was significantly decreased （P = 0. 000）. In- flammatory cytokines interleukin （IL） -1 beta and tumor necrosis factor-α （TNF-α） were highly ex- pressed in the model group, and the expression of IL - 1 beta and TNF-α could be effectively inhibited by p65 interference with RNA （P = 0.000）. Conclusion The specific interference of the expression of NF-κB p65 protein can effectively improve the pressure of tumor bearing and reduce the inflammatory re- sponse of mice under the subcutaneous implantation of glioma A172.