Study On Role Of Insulin In Pokemon Expression And Effect Of Pokemon In Cyclin D3 Transcription Regulation

Pokemon (POK erthroid myeloid ontogenic factor) is an oncogene expressed in many tissues and over expressed in tumors, it can regulate gene transcription. In this study, we designed the experiments to elucidate the mechanism of insulin induced Pokemon expression and mechanism of Pokemon down-regulated Cyclin D3 expression. Real-time PCR, Western blot, and Luciferase reporter assay were used to detect mRNA level, protein level, promoter activity, respectively. CHIP was carried out to test the protein-promoter interaction. It is showed that Pokemon mRNA and protein level was enhanced by insulin in a dose-dependent manner in HepG 2 cells. Using chemical inhibitors of insulin transduction pathways revealed that the increase of Pokemon mRNA and protein level was blocked by LY294002, a PI3K/Akt inhibitor. Insulin activated Pokemon promoter in a dose-dependent manner, while insulin did not affect the activity of Sp1 binding site mutated Sp1M promoter. These results suggest that insulin up-regulated Pokemon expression via PI3K/AKT pathway and transfactor Sp1. We also constructed various luciferase reporter constructs of Cyclin D3 5'-3'deletion fragment relative to a putative transcriptional start site. Pokemon decreased Cyclin D3 mRNA through Real-time PCR analysis. Transfection of the promoter constructs to HepG 2 and QGY 7703 cells showed Pokemon suppress Cyclin D3 promoter activity. CHIP analysis and GC-box mutated promoter experiment showed Pokemon binds to the GC-box (-14~-5bp relative to the putative transcriptional start site) to suppress Cyclin D3 expression. We demonstrated that HDAC1 bind to pLuc-375 through CHIP. So, HDAC1 may play a role in the transcriptional suppression of Pokemon. It is critical to understand Pokemon related transduction pathways and cross-talk for its important role in oncogenesis.