Small interfering RNAs targeting mutant K-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo

Abstract

Aim: 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.Materials and 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 Polyethylenimin e-encapsulated mutant K-ras targeted siRNAs (20 mg/mouse twice weekly) were effective in reducing tumor growth, when compared with controls (p < 0.05).Conclusion: 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.

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

“…Most studies dealing with K-ras silencing reported that siRNA or shRNA molecules could specifically inhibit mutant K-ras expression while leaving the WT K-ras unaffected although their sequences differed only in one nucleotide. [8][9][10][11]13 In our study, siRNA molecules that targeted K-ras mRNA outside the region with mutated codon reduced the levels of mRNA and protein of K-ras in both LoVo cells with mutated K-ras and in HT29 cells harboring WT K-ras. However, our results demonstrated that in HT29 cells, in which their oncogenic potential is due to other mutations, the effect of silencing K-ras did not reflect on cell survival.…”

“…As not every potential siRNA has an inhibitory effect, the choice of a proper siRNA sequence is crucial for efficient inhibition of K-ras expression. 12,13 Therefore, to identify the most effective silencing sequence, we tested three different siRNA-K-ras molecules, which resulted in different silencing effect. Differences in the efficacy of gene silencing with siRNA molecules might be associated with secondary structures at targeted position or with a mismatch at certain position, which could dramatically reduce the genesilencing effect.…”

“…The obtained results are consistent with the results from other in vitro studies performed mostly on pancreatic cell lines (Capan-1, Panc-1, MiaPaca-2 and PC-7), in which therapeutic potential of siRNA/ short hairpin RNA (shRNA) molecules directed against K-ras was also shown. [8][9][10][11][12][13] Furthermore, due to a relatively short half-life of synthetic siRNA molecules, 12,13,29,32 we constructed plasmid DNA that encoded a hairpin type of silencing molecules, that is, miRNA molecules with a sequence corresponding to the most efficient siRNA molecule (pmiRNA-K-ras). Most studies dealing with K-ras silencing reported that siRNA or shRNA molecules could specifically inhibit mutant K-ras expression while leaving the WT K-ras unaffected although their sequences differed only in one nucleotide.…”

“…In the experiments, in vivo, on pancreatic tumors, delay in tumor growth was observed. 11,13 Electroporation is a nonviral physical method for facilitated delivery of various molecules into cells, including genes. [14][15][16] Electrically assisted gene delivery, called electrotransfection, increased transfection efficiency of either reporter genes or therapeutic genes compared with injection of gene alone.…”

Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides

“…Among those point mutations, GAT, GTT and CGT mutation styles comprised more than 95% of the point mutations at codon 12 [18] . Therefore, K-ras point mutation at codon 12 is an early event for pancreatic cancer, which can be used as a target for early diagnosis and gene therapy [19,20] . K-ras gene mutation destroys the GTP enzyme activity of ras protein and makes K-ras active constantly, which makes K-ras protein unable to block signals for growth.…”