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

Abstract

Objective To investigate the inhibitory effects of RNA silencing via adenovirus-medi-ated β-catenin shRNA on proliferation of colon cancer ceils in vitro and in vivo.Methods Short hairpin RNA (shRNA) targeting β-catenin gene was designed, synthesized,and cloned into adenoviral expression vector AdHI-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 back-bone vector in 293A to pack the adenoviruses.The adenovirus titer was determined according to the plaque analysis method with a titer of 5 x 109 pfu/ml.HCT116 ceils 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.MTr 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 pAdHl-shβ-catenin-GFP group as compared with nor-real 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. Key words: Colon carcinoma; RNAi; βcatenin; Adenovirus