Investigation On Mechanism Of Pokemon And MiR-203Influencing The Biological Characters Of Human Glioma Cell Line U251

Background: Pokemon (POK erythroid myeloid ontogenic factor) is a new POKprotein family member that is located upstream of many proto-oncogenes and antioncogenes,which plays a critical role in the process of oncogene transformation. MicorRNA-203(miR-203) is a short, non-coding RNA that has been discovered recently. As othermicroRNAs, miR-203can regulate messenger RNAs (mRNA) of target genes by pairing withtheir3’UTR and affect their stability or direct their degradation to inhibit translation of targetgenes. Survivin is the smallest member of the inhibitor of apoptosis (IAP). The survivinprotein is expressed obviously in most human cancers and fetal tissue, but complete absencein terminally differentiated cells. Survivin can promote cell proliferation by affecting cellcycle and activity of caspase-3and caspase-7. The study has mainly investigated the influenceof pokemon and survivin on biological characters of human glioma cell line U251andclarified regulation mechanism to survivin.Objective: To observe the influence of pokemon and survivin on biological characters ofhuman glioma cell line U251and investigate regulation mechanism to survivin.Methods: U251cells were transfected with si-pokemon RNA, expression plasmid ofpokemon and mimics of miR-203using Lipofectamine2000, respectively. After transfection,cell growth, proliferation, apoptosis and sensitivity to TRAIL and celecoxib were determinedby growth curve, MTT assay and flow cytometry. The mRNA and protein level of survivinwas detected by Real-time PCR and Western blot. The ability of pokemon and miR-203wasconfirmed with dual-luciferase activity assay. Study on mechanism of pokemon bindingsurvivin promoter was demonstrated via chromatin immunoprecipitation assay.Results:1. The expression levels of pokemon and survivin in U251cells and glioma tissue wereobviously higher than HEB cells and normol brain tissu. After transfection of si-pokemonRNA in U251cells, growth curve and MTT assay revealed that growth and proliferationability of si-pokemon group were significantly suppressed,(P<0.05) and sensitivity to TRAIL and celecoxib was increased.Real-time PCR and Western blot showed that mRNA and proteinlevel of pokemon and survivin were down-regulated, respectively (P <0.05). On the contrary,expression level of pokemon and survivin were cooperatively up-regulated after pokemonexpression plasmid transfection (P <0.05). Dual-luciferase activity assay and chromatinimmunoprecipitation assay demonstrated that pokemon binds core promoter region ofsurvivin and activite its activity.2. The expression level of miR-203in U251cells was distinctly lower than normal glialcells HEB. Predicting outcomes of TargetScan indicated that survivin was target of miR-203.After over-expression of miR-203, Real-time PCR and Western blot showed that mRNA andprotein level of survivin were down-regulated, respectively (P<0.05). Dual-luciferase activityassay demonstrated that survivin was direct target of miR-203. Growth curve and MTT assayrevealed that growth and proliferation ability of miR-203group were significantly suppressed(P<0.05), and sensitivity to TRAIL and celecoxib was increased. U251cells transfectedmiR-203had a higher proportion of apoptosis than control group (P<0.01).Conclution: Protooncogine pokemon can promote growth and proliferation of U251cells, and weaken sensitivity to TRAIL and celecoxib; On the contrary, miR-203make U251cells display inversly biological characters, which is an “anti-proliferation and pro-apoptosis”miRNA. The mechanism of two kinds of phenomenon may be explained via regulatingexpression of survivin, this may provide a new method for exploring pathogenesis andbiological treatments of glioma.