腺病毒介导的RNA干扰对大肠癌HCT116细胞株β-连环蛋白表达的抑制作用

Inhibitory effects of adenovirus mediated β-catenin shRNA on proliferation of colon cancer cell line HCT116

目的构建针对人β-连环蛋白（catenin）的腺病毒载体pAdHl-shβ-catenin—GFP，应用RNA干扰（RNAi）的方法，观察其在体内和体外对人大肠癌细胞株HCT116生长的抑制作用。方法设计合成针对B—catenin的shRNA，并克隆至腺病毒表达载体AdH1-GFP上，制备针对β—catenin的腺病毒载体AdH1-GFP—shβ-catenin—GFP，与腺病毒骨架载体共转染293A细胞包装病毒，采用空斑法测定病毒滴度为5×10^9pfu／ml，感染HCT116细胞。应用Westernbolt和报告基因检测HCT116细胞B—catenin表达水平和转录活性，噻唑蓝（MTT）比色法测定活细胞数并绘制细胞生长曲线，建立软琼脂克隆形成实验观察肿瘤体外成瘤能力，同时建立裸鼠HCT116细胞移植瘤模型，观察肿瘤生长情况。结果成功构建针对β-catenin基因的RNAi腺病毒表达载体，与AdH1-GFP组比较AdH1-shβ、catenin—GFP组在感染24h和48hHCT116细胞β-catenin表达水平和转录活性显著降低，细胞的增殖和克隆形成能力则下降50％（P〈0．01）。体内实验表明重组腺病毒载体介导的β—cateninshRNA明显抑制肿瘤生长（P〈0．01）。结论腺病毒介导的RNAi技术可有效降低β—catenin在大肠癌细胞中的表达水平，抑制肿瘤细胞在体内外的增殖活性。

Objective To investigate the inhibitory effects of RNA silencing via adenovirus-mediated β-catenin shRNA on proliferation of colon cancer cells in vitro and in vivo. Methods Short hairpin RNA （shRNA） targeting β-catenin gene was designed,synthesized,and cloned into adenoviral expression vector AdH1-GFP to construct a pAdHl-shβ-catenin-GFP adenovirus vector containing green fluorescent protein （GFP） gene and expressing β-catenin shRNA. This plasmid was recombined with adenoviral backbone vector in 293A to pack the adenoviruses. The adenovirus titer was determined according to the plaque analysis method with a titer of 5 × 10^9 pfu/ml. HCT116 cells were infected with the recombinant adenovi- rus, and the expression of β-catenin as well as its transcriptional activity was examined using Western blot and luciferase reporter gene analyses. MTT and soft agar tumor formation assays were used to detect cell grow in vitro. The recombinant viruses were injected into the xenograft tumors of HCT116 cells in nude mice, and the tumor growth was observed. Results The recombinant adenovirus carrying shRNA targeting β-catenin had been constructed. Western blotting and luciferase assay showed a remarkable decrease of β- catenin expression and transcriptional activity in the pAdH1-shβ-catenin-GFP group as compared with normal control group. The tumor growth in pAdHl-shβ-catenin-GFP group was obviously slowed down, and the weight and volume of tumors were both significantly lower than those in the control group （ all P 〈 0.01 ）. Conclusion The shRNA targeting β-catenin constructed in the present study can efficiently decrease the β-catenin expression in HCT116 cells and suppress cell growth both in vitro and in vivo.