Inhibition Of Pokemon By ShRNA Mediated RNA Interference In Colorectal Cancer Cell SW480

ObjectiveTo investigate the expression and distribution of Pokemon in both 46 CRC tissues and SW480 cells. Construct siRNA expression vectors to explore the function of Pokemon during the metastasis of CRC by RNAi. The aim of the current study is to provide experimental basis for further research about the biological function of Pokemon in carcinogenesisMethods1. Tissues of CRC from 46 cases and the adjacent normal specimens, which were collected immediately after surgical resection. Semi-quantitative RT-PCR and immunohistochemistry were used to detect the expression of Pokemon at mRNA and protein levels, respectively.2. To construct three shRNA mediated eukaryotic expression plasmid of targeted against Pokemon gene.3. Semi-quantitative RT-PCR and immunofluorescence technology were applied to demonstrate the expression and localization of Pokemon in SW480 at mRNA and protein levels, respectively.4. The identified constructed plasmids were transfected into SW480 with LipofactamineTM2000. The expression of Pokemon mRNA and protein were detected with Real-time quantitative PCR and Western blot assay, respectively.5. Fluorescence Activated Cell Sorter was applied to study cell apoptosis of SW480 cells after transfection.Results1. The expression level of Pokemon mRNA in tumor tissues (0.845±0.174) was significantly higher than those in tumor adjacent specimens(0.321±0.087)(P<0.05). POKEMON protein was expressed in the cytoplasm and nuclear of cancer cells, with abundant positive signals in the cytoplasm. The positive ratio of Pokemon was 87 % in colorectal cancer tissues. In tumor adjacent specimens, Pokemon was expressed mainly in nuclear of glandular cells with faint positive staining and the positive ratio was 19.6 %. The positive expression ratio of POKEMON protein in CRC was significantly higher than that in tumor adjacent tissues (χ2=41.961, P﹤0.01).2. shRNA mediated RNAi plasmids were constructed correctly as proved by restriction analysis and by DNA sequencing.3. High expression of Pokemon mRNA was detected by RT-PCR in normal SW480 cells.Immunofluorescence staining showed that POKEMON protein was expressed in the cytoplasm and nuclear of cancer cells, with abundant positive signals in the cytoplasm.4. At 24 hours post-transfection, we found that the transfect efficiency is about 50%. At the same time, cell growths were obviously repressed and the characteristic morphology of transfected cells changes greatly against negative controls. The results of real-time quantitative PCR showed that the Pokemon mRNA expression levels have significantly degraded compared with the negative controls. The highest inhibitory rate of P2 is about 83.1%. The results of Western blot analysis showed that the POKEMON protein expression level has been significantly inhibited by 73.49%.5. Transfection with Pokemon siRNA expression vectors could induce cell apoptosis of SW480 cells.ConclusionsThe over-expression of Pokemon in CRC may play important roles in carcinogenesis and progressing of colorectal cancer. The shRNA mediated eukaryotic expression vectors targeting Pokemon gene were successfully constructed, which could effectively inhibite the expression of Pokemon at mRNA and protein levels. Transfection with Pokemon siRNA expression vectors could induce cell apoptosis of SW480 cells. The current results may be helpful in delineating molecular mechanism of Pokemon tumorigenicity and providing a potential route for CRC gene therapy in the future.