1.Downregulation Of TES By Hypermethylation In Glioblastoma Reduces Cell Apoptosis And Predicts Poor Clinical Outcome2.Differential Expressions Of DNMT3B Gene In GBM

Background: Glioblastmas (GBM) are the most common human brain tumors. arethe most aggressive, malignant and lethal tumor in brain. Median survival time of thistumor is only14.5months from diagnosis to death. Molecular abnormalities includegenetic and epigenetic defeats. Same as in other major tumors, globalhypomethylation and some region hypermethylation are hallmarker in thesemalignances, but reasons or clinical significance still remains unclear. To study DNAmethylation abnormality in GBM, we screened promotor region CpG islands on14475genes by using methylation specific array method with the expectation toidentify new therapeutic diagnostic marker to these lethal malignances.Methods: Normal and primary GBM (pGBM) tissues were collected from TiantanHospital. DNA from these tissues was analyzed with the DNA promoter methylationmicroarray. Protein expression was analyzed by immunohistochemistry, western blot,Cell apoptosis was detected by Flow cytometry analysis.Results: TES was hypermethylated in glioblastomas compared with normal braintissues using DNA promoter methylation microarray. The GBM patients with TEShypermethylation had a short overall survival (p <0.05, log-rank test). Among GBMsamples, reduced TES protein level was detected in33(89.2%) of all37tumor tissuesby immunohistochemical staining. Down regulation of TES was shown to becorrelated with tumor prognosis (p <0.05, log-rank test). After treated U251cell line with5-aza-dC, the expression level of TES was enormously up-regulated, and thencaused changes to the biological behaviors of cell lines and the apoptosis rate wasraised compared to the control group.Conclusions: This study demonstrated that hypermethylation of the TES geneoccurred at high frequencies in GBM, and there were significant correlations betweenTES hypermethylation and patients’ poor prognosis. TES expression in GBM cellswas found up-regulated after de-methylation treatment with5-aza-dC, and caused aninhibition of cell spread and increasing of their apoptosis. Our results suggest that theTES gene functions as a tumor suppressor gene which are mostly down-regulated inGBM patients by promotor hypermethylation, This study provides a new prognosticand therapeutic candidate target for GBM. Objective：Similar in other major malignances, DNA methylation abnormalities arebroadly exist in Glioblastoma(GBM), which include genome-wide globalhypomethylation and some promoter region hypermethylation, as promoter of tumordepressor genes. Reasons of these abnormalities are mostly remained unclear, alongwith clinical significances, as effects on tumor incidence, prognostic and therapeuticeffects, etc. DNA methylation is introduced on genome by DNA methyl-transferase1，3A，3B. Recent studies revealed that DNMT3a is mutated in about20%adult AMLpatients, but it is rare in other malignances, no report on GBM. This study wasscreened DNMT3B mutation in GBM patient samples with the expectation to find outreason(s) which responsible to genomic hypomethylation in these lethal malignances.Method：Total25GBM samples were examined in this study. Total RNA wasextracted from these patient samples. Full-length cDNA was amplified by RT-PCRand sequenced. Sequencing results from each sample were compared to NCBIpublished standard sequence by using DNASTAR software.Results：We successfully amplified DNMT3B from each patient samples. No pointmutation which alters amino acids was identified. But several splicing variants wereidentified in these patient samples which were differentially exited in the tumorsamples.Conclusion：no point mutation of DNMT3B was found in GBM patient samples. Twomajor DNMT3B splice variants (DNMT3B-V3and DNMT3B-V7) were founddifferentially exist in these tumor samples. Functions of these variants are subject tofurther elucidation.