靶向AKT1和COX-2的RNAi抑制U251胶质瘤细胞侵袭的体外实验

Knockdown of AKT1 and COX-2 by small interference RNA suppresses the invasion of malignant glioma U251 cells in vitro

目的应用RNA干扰(RNA interference,RNAi)技术敲低恶性胶质瘤细胞系U251中AKT1和COX-2表达后,在体外对细胞侵袭转移的抑制作用,初步探讨其可能的作用机制。方法构建重组腺病毒载体rAd5-A-C同时搭载靶向AKT1和COX-2的shRNA干扰序列,将其转染至恶性胶质瘤细胞系U251细胞。Realtime PCR检测RNAi后目的基因mRNA的表达水平,Western Blot检测目的基因及MMP-2、MMP-9、TIMP-2的表达情况。酶联免疫吸附试验检测转染前后分泌到细胞外的MMP-2和MMP-9的浓度变化。划痕实验和Transwell实验评价肿瘤细胞转染前后的细胞侵袭能力的变化。结果rAd5-A-C转染组AKT1和COX-2的mRNA和蛋白表达均明显抑制;MMP-2、MMP-9表达下调,TIMP-2表达则上调。ELISA检测胞外MMP-2、MMP-9浓度明显减低;划痕实验显示细胞转移运动能力明显减弱,Transwell体外侵袭实验结果显示穿过细胞数明显减低(P<0.001)。结论重组腺病毒介导的RNAi技术可以序列特异性地抑制U251细胞AKT1和COX-2的表达,在体外对U251细胞侵袭转移产生明显抑制作用,可能成为恶性胶质瘤治疗的新策略。

Objective To investigate the inhibition effects on tumor invasion of malignant glioma U251 cells by small hairpin RNA targeting AKT1 and COX-2 in vitro.Methods The recombinant adenovirus vector plasmid expression vector which contained short hairpin RNA （shRNA） expression construct of AKT1 and COX-2 was transfected into human malignant glioma U251 cells.AKT1 and COX-2 expressions were assessed using Real-time PCR and western blot analysis and the expressions of MMP-2,MMP-9 and TIMP-2 were detected by Western blot.The ectocytic density changes of MMP-2 and MMP-9 were detected using ELESA.The invasion ability of the tumor cells were measured by using Scarification and Transwell.Results Recombinant adenovirus vector rAd5-A-C mediated shRNA targeting AKT1 and COX-2 dramatically down-regulated the expressions of AKT1,COX-2,MMP-2 and MMP-9 in U251 cells and up-regulated the expression of,TIMP-2.The extracellular concentrations of MMP-2 and MMP-9 decreased in cell cultures transfected with shRNA targeting AKT1 and COX-2.Scarification indicated that the ability of invasion were decreased in cells transfected with shRNA targeting AKT1 and COX-2 compared with control group and nonsense sequence group.Transwell showed that the number of cells invading through the matrigel was significantly lower in rAd5-A-C transfection group （35.2±7.3） compared with control group （87±4.7）and nonsense sequence group （82±3.4）.Conclusions Knockdown of AKT1 and COX-2 by small interference RNA can significantly downregulate AKT1 and COX-2r expressions and inhibits the invasion ability of U251 cells in vitro.