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Construction of adenovector expressing small hairpin RNA targeting Bcl-XL and its anti-tumor effect
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Abstract:
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: \[99.0±26\]% vs \[ 99.0± 2.1\]%, 100% P<0.01), 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 \[ 9110± 389.1\] mm 3，P<0.01). 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.
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Project Supported:
Project supported by the National Natural Science Fundation of China（No. 30700970）
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