| CACX is one of the malignant tumors with high morbidity and bad prognosis, on which the current treatments such as surgical operation, radiotherapy and chemotherapy have shown very limited efficacy. Thus, it is necessary to develop new strategies to treat this type of carcinoma including biotherapy, immunotherapy and so on. Among of these strategies, gene therapy should be one of the potent candidates.RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies for virus infection, cancer and so on. For a successful cancer therapeutic strategy based on RNAi,the target of RNAi is critical since it can determine the therapy efficiency directly. Of course, the delivery system is critical too. Because the system is not only a competent vehicle to deliver intact siRNAs into target cells/organs in vivo, but also a nontoxic, nonimmunogenic, and low-cost vector. Importantly, off targeting remains a critical issue for therapeutic applications of RNAi and empirical tests for siRNAs with tolerable levels of off targeting are required.POKEMON is a member of the POK (POZ and Kru¨ppel) family of transcriptional repressors. As a central regulator of the important tumor suppressor ARF, Pokemon can specifically repress the transcription of the tumour suppressor gene ARF through direct binding. It was found that Pokemon aberrantly overexpressed in human cancers and that its expression levels predict biological behaviour and clinical outcome. All of these findings suggest that Pokemon may offer an effective new target for cancer therapeutics.In this study, RNAi strategy was be applied to develop potential method treating the CACX with the key oncogene Pokemon as target gene.Firstly,according to the mRNA sequence of Pokemon, 4 templates coding different siRNAs for Pokemon were designed and cloned in the pSilencer 5.1-H1 Retro vector,named the 4 recombinant plasmids HpsiRNA-1 to 4 respectively. The stable transfecting Hela cell lines were prepared by puromycin selection. the expression level of Pokemon gene in these Hela cell lines by Q-PCR which wsa normalized using the 2-△△CT method relatived to GAPDH, housekeeping gene, which was nearly equally expressed in all kinds of human cells. Then the reslut confirmed by western blot. To advestigate the relationship of Pokemon expression and the malignant phenotype of Hela cell lines, the proliferation ablity, the key apoptosis enzyme, Capase3/7 activity and the invasion ability of untransfected and stable transfecting cells were advestigated. The Q-PCR and WB results show that the expression of Pokemon gene in the cells transfected with HpsiRNA-4 recombinant plasmid is decreased to 83%, compared with the untransfected cells. And compared with the untransfected cells their proliferation value is about 65%, Capase3/7 activity is 2.2 times, invasion ablity was decreased to 1/3. The malignant phenotype of these cells had been weakening.To decreaseing the off targeting of RNAi and increasing the delivery efficiency, the HpsiRNA-4 recombinant plasmid was packaged with RGD-Poly K polypeptide to form the nanometer particles designated as"mimoretrovirus". Due to the characteristic of RGD to specifically binding with the integrin molecules on tumor cells and on nascent vascular endothelial cells, nano-particles containing RGD will directionally transport the recombinant retrovirus plasmids expressing siRNAs into tumor cells. Then the recombinant retrovirus plasmids integrated into the genome of tumor cells due to the property of retrovirus. The mimoretrovirus were prepared in microcentrifuge tubes by the addition of 1 ug of plasmid HpsiRNA-4 to serial dilutions of polypeptide in HBS. There are two methods to link RGD peptide and Poly K. one is through the peptide bond directly, named K18+RGD mimoretrovirus, and the other is through the linker, {acp}S{acp}S{acp}S, which can increase the flexibility,named K18+Linker+RGD. To facilitate our calculation of the peptide/DNA charge ratio, we assumed that each lysine in polypeptide carries one positive charge while each phosphate in DNA carries one negative charge. The peptide/DNA charge ratios were 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 respectively. To option the charge ratio, the complex was analyzed by electrophoresis on agarose gels and transmission electron microscopy analysis. To confirm the protection of the polypeptide, the DNase I protection assay was used. The results show No migration of the plasmid DNA band was found at the charge ratio of 2.0. This lack of migration indicated neutralization of the nucleic acid by the cationic peptide and/or formation of a large complex between the peptide and the plasmid DNA. The DNase I protection assay confirmed the result. When we used transmission electron microscopy with negative staining to investigate the mimoretrovirus, we found that the complexes formed a group of heterogeneous, dense particles with diameters about 20 nm at the peptide/DNA charge ratio of 2.0 and at the charge ratio of 4.0, the complex seemed closer. Then these particles including charge ratios from 0 to 8.0 were used to transfect the HpsiRNA-4 recombinant plasmid into Hela cells. As the control, we used the retrovirus,which titre are up to 105CFU/ml. the result shows that compared other charge tatio, when the charge ratio equaled 4.0, the expression of Pokemon gene decreased most. It was about downregulated to 43%, compared to untransfected cells. The silence effect obtained at this ratio was approximately equal to the level obtained by the liposome DOTAP-based technique. But it was lower than the retrovirus control, which could downregulate the Pokemon expression to 50%.Finally, we investigated the anti-tumor role of mimoretrovirus in vivo. We s.c. inoculated 1×107 of Hela cells into SCID mice to establish tumor-bearing mouse model for tumor prevention and treatment studies. In the prevention settings, mice were injected 4pmol of mimoretrovirus via vena caudalis for 4 times with 5-day interval at the time when they received the Hela cell grafting. At the ending time point of observation, no tumor developed in mimovirus group, recombinant retrovirus group and recombinant plasmid group, respectively. In contrast, the mice in control groups all developed tumor nodules that could be palpated 12 days post tumor cell inoculation. In the treatment settings, the animals were inoculated tumor cells and 12 days later injected with various therapeutic agents for the 1st time and followed by 3 times of sequentially repeated injections. Tumor growth in mice in group K18+RGD, K18+Linker+RGD and K18 was inhibited markedly, in comparason with the control mice. Among the therapeutic groups, the K18+Liner+RGD showed the highest anti-tumor effect.In summary, in this study we have successfully constructed the recombinant retrovirus-based plasmids, recombinant retrovirus and the mimoretrovirus, all expressing the siRNAs targeting human Pokemon gene. These therapeutic agents all could down-regulate the expression of Pokemon gene in tumor cell line and could reverse the malignant phenotype and biological features of the tumor cells. Furthermore, these effects could also be confirmed in a SCID mouse model. Tumor growth could be inhibited remarkablly in the tumor-bearing SCID mice when treated with these agents respectively. Howeve, among all the agents, the mimovirus showed the highest anti-tumor effectiveness, suggesting that not only Pokemon gene could be taken as a potential target for RNAi strategy, but also the mimovirus could be a novel and effective anti-tumor strategy that might be applied to other tumors and even to other non-tumor diseases. |
