论文部分内容阅读
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 inhibitory effect of AKT1 and COX-2 on the invasion and metastasis of human malignant glioma cell line U251 by RNA interference (RNAi) technology and to explore the possible mechanism. Methods Recombinant adenovirus vector rAd5-A-C was constructed and co-transfected with shRNA targeting AKT1 and COX-2. The shRNA was transfected into the malignant glioma cell line U251. Realtime PCR was used to detect the mRNA expression of target gene after RNAi. Western Blot was used to detect the expression of target gene and MMP-2, MMP-9 and TIMP-2. Enzyme-linked immunosorbent assay was used to detect the change of the concentration of MMP-2 and MMP-9 secreted outside the cell before and after transfection. Scratch assay and Transwell assay were used to assess the changes of cell invasiveness before and after transfection of tumor cells. Results The mRNA and protein expressions of AKT1 and COX-2 were significantly inhibited in rAd5-A-C transfected group. The expressions of MMP-2 and MMP-9 were down-regulated and the expression of TIMP-2 was up-regulated. The concentration of extracellular matrix metalloproteinase-2 and MMP-9 was significantly decreased by ELISA. The scratch test showed that the ability of cell migration was obviously weakened. The results of transwell invasion assay showed that the number of transmembrane cells was significantly decreased (P <0.001). Conclusions Recombinant adenovirus mediated RNAi technique can inhibit the expression of AKT1 and COX-2 in U251 cells in a sequence-specific manner, and significantly inhibit the invasion and metastasis of U251 cells in vitro. It may be a new strategy for the treatment of malignant glioma.