The Mechanism And Effect Of POK Erythroid Myeloid Ontogenic Factor (Pokemon) On The Proliferation And Apoptosis In Human Glioma Cells

BackgroundGlioma, the most common and low survival rate malignant tumor, existed in centralnervous system of adult. There is no clear boundary between glioma and surrounding normaltissues because the proliferation of glioma is invasive and unlimited. Glioma is harm topeople’s health due to low surgery cutting rate, high postoperative recurrence rate combinedwith poor therapeutic effect of chemotherapy and radiation therapy. POK erythroid myeloidontogenic factor (Pokemon) is highly expressed in many malignances. It has been suggestedthat Pokemon endows cancer cells with the capability of anti-aging and anti-apoptosis, andcan also regulate the activity of a wide range of oncogenic genes and tumor suppressor genes.Based on these findings, Pokemon has been proposed to function as a switch of tumorgenesis.Therefore, Pokemon has the potential to become a new target for the gene therapy of tumors,its expression and biological characteristics was not proven in glioma. Survivin is animportant oncogene of apoptosis inhibitory protein family, which over-express in glioma. Itcan promote cell growth and obtain the anti-apoptosis ability by blocking cell cycle andinhibiting activity of caspase-3and caspase-7. Cell cycle protein B1(CyclinB1) is a cell cycleprotein coded by CCNB1gene, which can regulate the cell cycle of eukaryotes effectively,especially in the beginning, middle and later stage of mitosis in glioma cell, as well as exitregulation of mitosis. However the regulation and effect of Pokemon on Survivin andCyclinB1gene expression in glioma cells still remains unclear. The aim of the study mainly isto investigate the mechanism and effect of Pokemon on the expression of Survivin andCyclinB1, by which Pokemon promote cell proliferation and survival in glioma U251cells. PartⅠThe Expression and Role of Pokemon in Malignant GliomaOBJECTIVE To investigate the Pokemon expression in human glioma tissues， humanglioma U251cells and glial HEB cells, and to analyze the relationship between theexpresseion of Pokemon and proliferation and differentiation in human glioma.METHODS Immunohistochemical staining, RT-PCR and western blotting was used todetect the expression of Pokemon in35cases of glioma tissues, and18cases of normal braintissue in comparison with operation excision, and human glioma U251cells and glial HEBcells. The32cases of glioma tissue from operation excision were pathologically identifiedand classified according to the WHO standards. Cell counting, MTT assay and colonyformation test were employed to determine the proliferation of glioma U251cells.RESULTS The positive rate of6normal brain tissues was16.7%(1/6). The positive rate ofPokemon expression in35glioma tissues was82.8%（29/35, of them, that of groupastrocytoma Ⅰ, Ⅱ, Ⅲ and Ⅳ were respectively60.0%,76.9%,83.3%and100.0%, andpositive rate of cells in positive tissues were13±8.5％,28.6±10.6％,44.5±7.5％and59.7±17.9％, respectively. The expression level of Pokemon gradually increased followingpathologic grading of glioma, which was positively correlated by Spearman rank level rank (r=0.514, p<0.01).The expression level of Pokemon mRNA and protein in glioma tissues orU251cells was higher than that in normal brain tissues or HEB cells(p<0.05). Pokemonsilencing could significantly suppress the proliferation (p<0.05) and reduce the colonyformatting in U251cells(p<0.05).CONCLUTION The expression of Pokemon was present in glioma tissues, and normalbrain tissues, and U251cells and HEB cells. The expression level of Pokemon both mRNAand protein in glioma tissues or U251cells was higher than that in normal brain tissues orHEB cells. Pokemon expression could be used as the marker of pathologic grading of glioma.Pokemon induced the proliferation and colony formatting in U251cells. PartⅡ Mechanism and Effect of Pokemon on Expression ofSurvivin in GliomaOBJECTIVE To investigate the mechanism and effects of Pokemon on expression ofSurvivin and to explore the relationship between survival and Survivin expression inducedby Pokemon in U251cells.METHODS The expression of Survivin was detected by RT-PCR and western blotting inglioma tissues, and normal brain tissue, and human glioma U251cells and glial HEB cells.The proliferation was determined by cell counting and MTT assay in glioma U251cellspre-incubation with or without TRAIL and celecoxib. The effect of Pokemon on the activityof Survivin promotor was analyzed by chromatin immunoprecipitation (ChIP) and the dualluciferase reporter gene assay.RESULTS The expression of Survivin was present in glioma tissues, and normal braintissues, and U251cells and HEB cells. The expression level of Survivin both mRNA andprotein in glioma tissues or U251cells was higher than that in normal brain tissues or HEBcells (p<0.05or p<0.01). Pokemon silencing could significantly reduce the Survivinexpression (p<0.05) and statistically increase the sensitivity of U251cells to both TRAIL andcelecoxib (p<0.05). The overexpresion of Pokemon could significantly induce the expressionlevel of Survivin, which was time-and dose-dependent, and heighten the drug resitance toboth TRAIL and celecoxib and survival in glioma U251cells. Pokemon bound to the coreseguence(+84~-396bp) of Survivin promoter and activated Survivin promoter.CONCLUTION The expression of Survivin was present in glioma tissues, and normalbrain tissues, and U251cells and HEB cells. The expression level of Survivin both mRNAand protein in glioma tissues or U251cells was higher than that in normal brain tissues orHEB cells. Pokemon induced the expression and increased the drug resitance to both TRAIL and celecoxib and survival in glioma U251cells. Pokemon bound and activated promoter ofSurvivin.Part Ⅲ Mechanism and Effect of Pokemon on Expression ofCyclinB1in GliomaOBJECTIVE To investigate the mechanism and effects of Pokemon on expression ofCyclinB1, and to explore the relationship between proliferation and Survivin expressioninduced by Pokemon in U251cells.METHODS The expression of CyclinB1was detected by RT-PCR and western blotting inhuman glioma U251cells and glial HEB cells. The proliferation was determined by cellcounting and MTT assay in glioma U251cells pre-incubation with or without Daunorubicin.Cell cycle was analyzed by flow cytometry. The effect of Pokemon on the activity ofCyclinB1promotor was analyzed by the dual luciferase reporter gene assay.RESULTS The expression of CyclinB1was present in both glioma U251cells and glialHEB cells. The expression level of CyclinB1mRNA in U251cells was higher than that inHEB cells (p<0.05). The overexpression of CyclinB1significantly increased the expressionlevel of CyclinB1(p<0.05), and Pokemon silencing reduced the expression level of CyclinB1(p<0.01), which was time-and dose-dependent. Pokemon activated promoter of CyclinB1.Pokemon silencing significantly inhibited proliferation (p<0.05), blocked at G2/M phase incell cycle, and statistically heightened the drug sensitivity to Daunorubicin in U251cells(p<0.05).CONCLUTION The expression of CyclinB1was present in U251cells and HEB cells. Theexpression level of CyclinB1in U251cells was higher than that in HEB cells. Pokemoninduced expression of CyclinB1by activating the promoter of CyclinB1. siRNA-CyclinB1 blocked at G2/M phase in cell cycle, and statistically heightened the drug sensitivity toDaunorubicin in U251cells.CONCLUTION1. The Pokemon, CyclinB1and survivin was significantly overexpressed in human gliomaand U251cells. Pokemon expression could be used as the marker of pathologic grading ofglioma.2. Pokemonin induced the expression of both CyclinB1and survivin by activating thepromoter of CyclinB1and survivin, respectively.3. The overexpression of Pokemonin induced the expression of both CyclinB1and survivin,and heightened the survival and stimulated proliferation in U251cells.