| BackgroundMalignant astrocytic gliomas such as glioblastoma are the most common andlethal intracranial tumors. These cancers exhibit a relentless malignant progressioncharacterized by widespread invasion throughout the brain,resistance to traditional andnewer targeted therapeuticapproaches, destruction of normal brain tissue, and certaindeath. The recent confluence of advances in stem cell biology, cell signaling, genomeand computational science and genetic model systems have revolutionized ourunderstanding of the mechanisms underlying the genetics,biology and clinical behaviorof glioblastoma.This progress is fueling new opportunities for understanding thefundamental basis for development of thisdevastating disease and also novel therapiesthat, for the first time, portend meaningful clinical responses.Malignant gliomas are classified and subtyped on thebasis of histopathologicalfeatures and clinical presentation. The most common and biologically aggressive ofthese is glioblastoma (GBM), World Health Organization (WHO) grade IV, and isdefined by the hallmark features of uncontrolled cellular proliferation, diffuseinfiltration,propensity for necrosis, robust angiogenesis,intense resistance to apoptosis,and rampant genomicinstability. As reflected in the old moniker “multiforme,”GBMpresents with significant intratumoral heterogeneity on the cytopathological,transcriptional, and genomic levels. This complexity, combined with a putative cancerstem cell (CSC) subpopulation and an incomplete atlas of (epi)genetic lesions drivingGBM pathogenesis,has conspired to make this cancer one of the most difficult tounderstand and to treat. Despite implementation of intensive therapeutic strategies andsupportive care, the median survival of GBM has remained at12month over the pastdecade. Populations of stem cells with unique properties exist in essentially all tissues ofmulticellular adult organisms. These cells are capable of self-renewal and can give riseto all cell lineages of the tissue they reside in. Stem cells are long-lived, being presentthroughout the entire life span of organisms; a characteristic that made them attractivetargets of tumorinitiating transforming events that require years toaccumulate.According to the “stem cell origin of cancer”hypothesis, stem cells or cellsthat acquired the ability to self-renew accumulate genetic changes over long periods oftime, escape from the control of their environment, and give rise to cancerous growth.This distinct cell of origin may explain the well-known inter-tumoral heterogeneity ofbreast and other carcinomas. However, most tumors are largely composed of cells withsome degree of differentiation, based on which the tissue of origin can be determinedeven in the case of distant metastases. One of the postulations of the “cancer stem cell”hypothesis is that a opulation of cells with stem cell-like features exists in tumors andthis population gives rise to the bulk of the tumor cells with more differentiatedphenotypes.Our study investigated the expression of AMF in U87SP cells.Furthermore,wedown regulated the AMF in U87cells and evaluated the influence to the migrationability and tumor sphere forming.At the same time,our study also investigate thepotential association between EGF+61G/A genotype and brain glioma in a Chinesepopulation.So the role of AMF is important in stemness of U87cell,it may be a newspecial marker of CSC in future.Part1:The expression of AMF in cancer stem-like cells in the U87glioma cell lineObjective:To investigate the expression of AMF in cancer stem cells of U87.Methods: The U87glioma cells were enrichment cultured through serum freemedium.FACS was used to analysis the ratio of SP cells and separate the SPcells.Western blot was used for investigate the expression of CD133,SOX2and AMF inSP cells.Results: When the U87was cultured in traditional serum supplement edmedium,U87cells grew as single cells attached to the culture flask,while presented assingle cells suspending in medium after reseeded in serum-free neural stem cellmedium，as small tumor spheres would formed in48hours.The growth style may switchin two kinds of mediums.SOX2、CD133and AMF only high expression in SP cells,P＜0.05. Conclusion:1.The fraction of SP cells in U87cell line is0.5%.2.Nerual stem cellserum free medium can enrichment CSC in U87cell line.3.AMF is high expression inSP cells.Part2:Downregulation of Phosphoglucose Isomerase/Autocrine MotilityFactor Causes inhibition of migration ability and tumor sphere forming in U87celllineObjective:To investigate the expression of PGI/AMF and related mechanismsinvolved in glioblastoma migration and the stemness of glioblastoma.Methods: siRNA was used for the knockdown of AMF in U87;the wound healingassays and transwell assays was used for evaluated the immigration ability of U87.Theratio of SP cells in U87cell was evaluated by FACS analysis.Results: siPGI/AMF exhibited decreased expression of endogenous PGI/AMF atprotein level>70%, respectively, by Western blot analysis. Interestingly,down-regulation of endogenous PGI/AMF by siRNA led to the complete depletion ofPGI/AMF secretion;In siPGI/AMF cells, the wound healing was inhibited in contrastwith parental;siPGI/AMF cells showed decreased transmigration ability(p=0.0005);ThesiPGI/AMF cell formed50%fewer tumor spheres compared with the number of tumorspheres formed by the control cells (p=0.0019). In addition, the size of the tumorspheres was also significantly reduced in cells knocked formed by control cells was197.3±8.090μm compared with113.0±10.02μm for the spheres formed by siPGI/AMFcells (p=0.0028);Knockdown of PGI/AMF in U87cells downregulates Akt pathway andstemness-related proteins.Conclusion:Our data confirm the functional importance of PGI/AMF in themigration ability of GMB cells. Glioma cells with reduced expression results in cellswith reduced migration ability and reduced ability to form tumor spheres. Theseobservations, together with the gene expression analysis data, highlight the role ofPGI/AMF in glioma migration and stem cell biology.Part3:A Functional Polymorphism in the Epidermal Growth Factor Gene IsAssociated With Risk for Glioma in a Chinese Population Through Regulation ofAMFObjective:The purpose of this study was to investigate the potential associationbetween EGF+61G/A genotype and brain glioma in a Chinese population.Methods: PCR-RFLP was used for the evaluate the genostype of EGF+61，theexpression level of AMF was investigated by RT-PCR. Results: Characteristics of the180patients with glioma and180controls areshown in Table1. The groups did not differ regarding sex (p=0.50) or age (p=0.52).The mean age–standard deviation was41.5–12.5years for the glioma group and40.8–11.7years for the control group. In addition, the2groups did not differ forsmoking status, drinking, or family history of cancer. Among the180patients withglioma,67(37.2%) had astrocytomas,59(32.8%) had glioblastoma, and54(30.0%) hadother gliomas. Genotype and allele frequencies were in Hardy-Weinberg equilibrium inboth groups.Patients with glioma had a significantly higher frequency of AA genotype(OR,2.25;95%CI,1.20,4.21; p=0.01) than did controls (Table2). When stratified byhistologic and World Health Organization grade of glioma, the distribution of eachgenotype did not significantly differ between groups.Conclusion:Our data suggested that the EGF+61AA genotype was associatedwith a higher glioma risk in a Chinese population. This is in contrast with previousstudies,which reported G allele as a risk factor of glioma in white persons. Additionalstudies are needed to confirm this finding. |
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