Construction of adenovector expressing small hairpin RNA targeting Bcl-XL and its anti-tumor effect

Objective: To construct the adenovector expressing small hairpin RNA targeting Bcl-XL(Ad/Bcl-XL shRNA),and evaluate its anti-tumor effect.Methods: Firstly,Ad/Bcl-XL shRNA was constructed and purified.Then the protein level of Bcl-XL and survival of colon cancer cells after the treatment of Ad/Bcl-XL shRNA were determined by Western blotting and MTT assay,respectively.Furthermore,the activation of apoptotic signaling was also detected by Western blotting assay.Finally,the anticancer effect of Ad/Bcl-XL shRNA in vivo was confirmed in the subcutaneous tumor model derived from DLD1 cells in nude mice.Results: Ad/Bcl-XL shRNA was constructed and purified successfully.It obviously down-regulated the Bcl-XL protein and significantly inhibited the growth of DLD1 cells(1 000 MOI and 2 000 MOI Ad/Bcl-XL shRNA group was(MOI=1 000: [60.6±4.8]% vs [37.3±6.9]%,100%;MOI=2 000: [99.0±2.6]% vs [99.0±2.1]%,100% P0.01),but had no obvious toxicity on normal human fibroblasts.Western blotting results demonstrated that the apoptotic signal molecules including caspase-9,caspase-3,and PARP were obviously activated after the treatment with Ad/Bcl-XL shRNA.In vivo,it also dramatically suppressed the growth of subcutaneous tumors derived from DLD1 cells in nude mice(eg.29th day Ad/Bcl-XL shRNA group was [250.1±185.7] vs Ad/GFP [880.0±286.1],PBS [911.0±389.1] mm3,P0.01).Conclusion: Ad/Bcl-XL shRNA can down-regulate the expression of Bcl-XL and inhibit the growth of colon cancer cells in vivo and in vitro,suggesting that it may be a new strategy to treat the colon carcinoma.