Expression of the Proto-oncogene Pokemon in Colorectal Cancer - Inhibitory Effects of an siRNA

Objective: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenicfactor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA)expression vector in SW480 and SW620 cells.
Methods: Semi-quantitative reverse transcription-polymerasechain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expressionlevels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the locationof Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a shorthairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expressionlevels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and westernblot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated withreference to growth curves with MTT assays.
Results: The mRNA expression level of Pokemon in tumor tissues(0.845 ± 0.344) was significantly higher than that in adjacent tumor specimens (0.321 ± 0.197). The positiveexpression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues(19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 andSW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5%at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively.MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells.
Conclusion:Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expressionvectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, therebyreducing malignant cell proliferation.