Reversal of the phenotype by K-rasval12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1.
OBJECTIVE
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-ras(val12) 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-ras(val12). After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-ras(val12) was obtained. Stable suppression of K-ras(val12) was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry.


RESULTS
We obtained adenovirus AdH1-K-ras(val12) carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-ras(val12), 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-ras(val12)-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).


CONCLUSIONS
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-1 can be induced after AdH1-K-ras(val12) infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.
Chen
Li-Mo
LM
Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Le
Huang-Ying
HY
Qin
Ren-Yi
RY
Kumar
Manoj
M
Du
Zhi-Yong
ZY
Xia
Rui-Juan
RJ
Deng
Jing
J
D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D011905:Genes, ras / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
D034741:RNA, Small Interfering