AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism.METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-rasval12 and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-rasval12. After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-rasval12 was obtained. Stable suppression of K-rasval12 was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry.RESULTS: We obtained adenovirus AdH1-K-rasval12 carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-rasval12, the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-rasval12-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection)compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%,3.78% at 72 and 96 h post-infection of AdH1-p53,respectively) (P＜0.05).CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi)to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1can be induced after AdH1-K-rasval12 infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.