Small interfering RNAs targeting mutant K-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo.
OBJECTIVE
To investigate the effect of small interfering RNAs targeting mutant K-ras on the growth of pancreatic carcinoma cell lines in vitro and in vivo.


METHODS
We cloned targeting sequence spanning codon 12 of mutant K-ras into the pSilencer-hygro plasmid, yielding two recombinant vectors with one base different. Both human pancreatic carcinoma cell lines were transfected by these two recombinant vectors. The transfected PC-7 cells were injected subcutaneously into nude mice to observe its tumorigenicity. RT-PCR and Western blot analysis were carried out to test the expression of K-ras in all of the transfected cell lines. Growth curves assay were performed to test the abilities of cells proliferation. Anti-K-ras therapy of PC-7 and Panc-1 in subcutaneous mice models were performed by intratumor injection of polyethylenimine/siRNAs complex.


RESULTS
The expressions of K-ras in PC-7 cells and Panc-1 cells were significantly inhibited by corresponding small interfering RNAs. The expression of K-ras was particularly inactivated by siRNA without any base mismatch to its homologous mRNA, while this oncogene with central base mismatch could not be inhibited as effectively as that of the former. The growth of PC-7 cells and Panc-1 cells transfected by corresponding mutant K-ras targeted siRNAs were significantly suppressed when compared with controls (p<0.05). The transfected PC-7 cells lost tumorigenic ability. Four weeks treatment of Xenograft of pancreatic carcinoma (PC-7 and Panc-1) in nude mice with Polyethylenimine-encapsulated mutant K-ras targeted siRNAs (20 microg/mouse twice weekly) were effective in reducing tumor growth, when compared with controls (p<0.05).


CONCLUSIONS
The central base may play a key role in the process of RNA interference. The mutant point and its vicinity of 19 nucleotides in K-ras may be the effective targeting sequence for RNA interference. Targeting mutant-k-ras therapy of pancreatic carcinoma may be a clinically applicable therapeutic modality.
Zhu
Hong
H
Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
Liang
Zhi Yong
ZY
Ren
Xin Yu
XY
Liu
Tong Hua
TH
D002455:Cell Division; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D017931:DNA Primers; D020868:Gene Silencing; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics* / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D034741:RNA, Small Interfering