The Expression Of GBE1 In Human Brain Glioma And Intervention Research

Glioma is the most common primary brain tumor, and represents about 40% of all brain tumors, with a 5- year survival rate of 20-30%. Among gliomas, glioblastoma multiforme(GBM) accounts for >50% and is the most malignant pathological type with a yearly incidence of 3/100,000-4/100,000. C haracterized by diffuse infiltration, GBM is known for high recurrence and poor prognosis, with a median survival of 14.6 months and a disillusioning 5-year survival rate of only 2%. Despite advances in treatment approaches and strategy, the median survival time and overall survival in such patients remain poor without significant improvement in the last decade. Therefore, further investigation of molecular mechanisms in initiation and progression of glioma and exploration of key molecular targets will be helpful to break through the bottleneck of treatment and improve glioma prognosis.Reprogramming of energy metabolism is one of the major cancer recognition in the last decade, and is considered as one of the ten hallmarks of cancer, which including sustaining growth and proliferation signaling, insensitivity to growthsuppressors, resisting cell death, replicative immortality, inducing angiogenesis, activating invasion and metastasis, evading immune destruction, tumor-promoting inflammation, genome instability and mutation. As the essential factor in maitenance of cell survival, energy is particularly important for cancer cell to sustaining growth and proliferation. Energy metabolism is therefore speculated to the Achilles’ heel of cancer, and targeting it as a breakthrough might be the most effective strategy to fight against cancer. As the most important energy resources, glycose metabolism undoubtedly attracts more attention in this fight, and its related enzymes are thought to be the key point of attack.GBE1(glucogen branching enzyme 1) protein molecule, one of α-amylase family members, is a key enzyme in glycogen synthesis with the function to transfer glycose chain and form glycogen branches. It is usually regarded that the highly-branched structure of glycogen exerts two functions: first, it increases the solubility of glycogen and facilities the storage in cytosol; and secondly, it provides a multitude of docking sites for glycogen-binding proteins, including related enzymes and regulatory proteins in glycogen metabolism. Glycogen, the major energy storage form in cytosol, has been found to be highly accumulated in many tumors such as colorectal cancer, gastric cancer, thyroid cancer, and glioma. Glycogen can be fast hydrolyzed and provide energy to maintain survival of cancer cell in response to impoverishment of nutrients and oxygen of the microenvironment. In addition, it is also reported that glycogen metabolism plays an important role in post-translational modification, biosynthesis and antioxidant defences. Disruption of glycogen metabolism in cancer cell has been documented to arrest cell cycle and induce apoptosis through reducing activity of pentose phosphate pathway, inhibiting biosynthesis of nucleic acid and lipid, and increasing levels of reactive oxygen species. Therefore, we speculated whether inhibition of glycogen synthesis through GBE1 could be an effective way to treat glioma.This dissertation systematically expounded the significance and expression of GBE1 in human brain glioma, analyzed the effect of GBE1 RN A intervention on biological behaviors, and discussed the related molecular mechanisms.1. Expression and significance of GBE1 in human gliomaIn this part, immunohistochemical staining, western blotting and RT-PCR were used to detect expression level of GBE1 protein and m RNA in 118 cases of human glioma tissues and 10 cases of normal brain tissue. Then, the difference in various pathological types and grades were analyzed. O verall, GBE1 expression level was low in normal brain tissues, while it increased significantly in glio ma tissues with expression in both nucleus and cytosol(p<0.05). Statistical results according to WHO classification and pathological types showed that, except for the WHOI and pilocytic astrocytoma, the rest of grades and types generally over expressed GBE1 protein(p<0.05), and there was a linear correlation between GBE1 expression and tumor malignant grades(p<0.05).A follow-up of 118 cases of glioma patients was conducted, and the relationship between GBE1 protein level and clinical data was then analyzed. Results of Mann-Whitney rank test indicated that IRS of GBE1 protein was not associated with gender, age, and tumor size. The Kaplan-Meier survival analysis showed that the median survival of glioma patients with lower GBE1 protein expression and those with higher GBE1 expression was 14 and 33 months respectively. Furthermore, Log-rank test indicated shorter survival in glioma patients with higher GBE1 IRS(p<0.05). The multivariate Cox regression analysis found that pathological grade and IRS of GBE1 protein were two independent factors affecting the survival of glioma patients(p<0.05). In addition, four GBMs gene expression datasets from TC GA and GEO database were used to evaluate GBE1 m RNA expression level and clinical significance. As a result from all 4 datasets, GBE1 m RNA expression level was higher in GBM than normal brain tissue(p<0.05). Kaplan-Meier survival analysis indicated that the median survival of GBM patients with higher GBE1 m RNA expression is shorter than that of patients with lower GBE1 m RN A expression(p<0.05). Multivariate Cox regression analysis showed that MGMT promoter methylation status, GBE1 m RNA expression and patients’ age were independent prognostic factors in GBM patients. These results suggested that comprehensive evaluation of GBE1 m RN A expression, MGMT promoter methylation status, pathology and age should be helpful to predict prognosis of glioma patients.2. The effect of GBE1 RNAi on biological behaviors of glioma cellsIn this part of study, human glioma cell line U251 was chosen as research subject, and the influences of down-regulation of GBE1 expression by RNAi technology on cell proliferation, apoptosis, migrative and invasive ability in U251 cells were detected. Results from MTT assay and plate colony formation assay showed that, cell viability and colony formative ability were significantly inhibited in U251 cells treated by GBE1 RNAi(p<0.05). Flow cytometry was performed to detect the effects of GBE1 down-regulation on cell cycle progression and apoptosis. Compared with control cells, U251 cell treated by GBE1 RNAi were obviously arrested in S phase and induced more apoptosis(p<0.05). Moreover, wound healing assay and matrigel-transwell assay showed that GBE1 RNAi significantly inhibited the migration and invasion of U251 cells in vitro. Taken together, it was suggested that GBE1 may play a certain role in the development and progression of glioma, and interfering GBE1 as a target might inhibit glioma cell proliferation and invasion in certain degree.3. Possible molecular mechanisms of GBE1 influencing biological behaviors of glioma cellWestern blotting analysis was performed to detect the protein level of apoptosis- and invasion-related markers in U251 cells treated by GBE1 RNAi. As a result, protein level of pro-apoptosis factor Bax and claved-caspase3 was significantly increased(p<0.05), whereas anti-apoptosis factor Bcl-2 protein level was apparently decreased(p<0.05). Moreover, the expression of invasion-related factor MT1-MMP was also enhanced(p<0.05), but MMP-2 protein level was not found to be significantly alterated(p>0.05). Results from gene expression correlation analysis based on TCGA GBM dataset, there were significant correlations between the m RN A expression levels of GBE1 and other important proteins such as beta-catenin, c-MYC, MT1-MMP, HIF-1α, VEGF and GYS respectively(p<0.05). Western blotting and immunocytochemical methods were further conducted to investigate the relationship between GBE1 and beta-catenin. It was found that down-regulation of GBE1 expression not only reduced beta-catenin protein level in the cytosol, but also suppressed its translocation to nucleus in U251 cells. It wasspeculated that inhibition of beta-catenin transcription level maybe one of the mechanisms of GBE1-RNAi influencing cell biological behaviors of U251 cells. Finally, western blotting and RT-PCR were used to detect the expression change of GBE1 in glioma cell lines in response to hypoxia. The results showed that, under hypoxic environment, the protein level and m RNA expression of GBE1 was increased remarkably in all of U251, U87 MG and SHG44 cell lines(p<0.05).In summary, the above results suggested that GBE1 might play important promotive roles in the development and progression of human brain glioma. GBE1 was speculated to be not only a prognostic factor of glioma patients, but also a potential therapeutic target, providing a new choice for glioma personalized treatment.