Study On The Antitumor Efficacy And Mechanism Of Short Interfering RNAs

The overexpression of oncogenes are genomic features of human cancers. Inhibition of abnormal expression of these oncogenes has been a common idea for development of new antitumor drugs. It has been identified that RNA interference technology is a more powerful tool than antisense technology for silencing endogenous or exogenous genes in mammalian cells. When the 21-25 bp exogenous short interfering RNA (siRNA) was transfected into mammalian cells, the siRNAs specifically recognized and bound to the homologous sequences of mRNA of target genes, and the mRNA was cut at the central section of the complex composed of siRNA, mRNA and endogenous nucleases. In the present study, we selected some tumor-related genes as targets to design and synthesize siRNAs against mRNAs of target genes. Furthermore, we screened promising antitumor drugs from them and investigated the antitumor efficacy and molecular mechanisms of these siRNAs.Carcinogenesis is regulated by network of many genes associated with apoptosis, proliferation, cell cycle progression and etc. We synthesized siRNAs against mRNAs of tumor-related genes, including bcl2, cdk2, hras, pkc-a, mdm2 and vegf. These siRNAs were transfected into various tumor cells with liposome, and cytotoxicity of these siRNAs was determined with MTT assay. The results showed that the IC50 values of bcl2-siRNA,mdm2-siRNA,cdk2-siRNA,pkca-siRNA, hras-siRNA and vegf-siRNA were 196.7, 137.7, 191.5, 260.1, 187.0 and 2416.7 nM, for human breast cancer MCF-7 cells, respectively. The IC50 of mdm2-siRNA was 137.7 nM for MCF-7 cells, 240.3 nM for human hepatoma BEL-7402 cells, 332.3 nM for human oral epithelial carcinoma KB cells and 1404.5 nM for human colon carcinoma HT-29 cells. The data showed that the cytotoxicity of mdm2-siRNA was 10-fold higher to p53 wild-type MCF-7 cells than to p53 mutant HT-29 cells, indicating that mdm2-siRNA-mediated cytotoxicity was dependent of p53 signaling pathway. We chose mdm2-siRNA as object for further research.We assessed the silencing effect of mdm2-siRNA on its target mRNA by RT-PCR. The results showed that mdm2 mRNA was markedly down-regulated in MCF-7 cells transfected with 100 nM mdm2-siRNA for 24 h, while mock-siRNA was ineffective,...