Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells

Aim:RNA interference(RNAi)has been proposed as a potential treatment forcancer,but the lack of cellular targets limits its use in cancer gene therapy.Nocurrent technology has achieved direct tumor-specific gene silencing using RNAi.In the present study we attempt to develop a tumor-specific RNAi system usingthe human telomerase reverse transcriptase(hTERT)promoter:furthermore,weanalyzed its inhibitive effect on Bcl-2 expression.Methods:The vectors contain-ing a small hairpin RNA(shRNA)to target exogenous reporters[firefly luciferaseand enhanced green fluorescent protein(EGFP)]and endogenous gene(Bcl-2)were constructed.Luciferase expression was determined by dual luciferase assay.Reverse transcription-polymerase chain reaction(RT-PCR),fluorescence micros-copy and fluorescence-activated cell sorting(FACS)were used to measure EGFPexpression,Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting.Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.FACS was used to analyzethe cell cycle distribution profile.Results:We showed that with the hTERTpromoter directly driving shRNA transcription,expression of the exogenous re-porters(LUC and EGFP)in tumor cells,but not normal cells,was specificallyinhibited in vitro.The hTERT promoter-driven shRNA also depressed the expres-sion of Bcl-2.Inhibition of Bcl-2 did not affect cell proliferation,but increased thechemosensitivity of HeLa cells to 5-fluorouracil.Conclusion:The present studydescribes an efficient RNAi system for gene silencing that is specific to tumorcells using the hTERT promoter.Suppression of Bcl-2 by using this system sen-sitized HeLa cells to 5-fluorouracil.This system may be useful for RNAi therapy.