| Glioblastoma multiforme is one of the most common clinical brain tumors. With the continuous advancement of technology and progression of medicine, the diagnosis and treatment of gliomas have achieved great success, but the patient’s prognosis has not been significantly improved. Identification of mutated genes in glioblastoma multiforme (GBM) is an essential step towards to a better understanding of the molecular mechanisms of this disease, establishing new targets for its diagnosis and therapy.Myeloid leukemia cells-1 (Mcl-1) protein is a member of the Bcl-2 antiapoptotic protein family, which is involved in the regulations of cell cycle, cell apoptosis as well as cell differentiation. Excessive expression of Mcl-1 in cells can cause the occurrence of malignant tumor, whereas inhibiting Mcl-1 protein expression can not only promote the apoptosis of tumor cells also can increase the sensitivities of the tumor cells to chemotherapy and radiotherapy. When combineing with antiapoptotic proteins, Mcl-1 can inhibit their antiapoptotic activity. The Mcl-1 mutation can protein’s half-life can get changed, so as to prolong the antiapoptotic effect. It may also be a possible mechanism for the occurrence of glioma.By direct sequencing, we screened 20 genes which were related to malignant tumors, including Mcl-1, Bcl-2, EGFR ect. as candidate genes. By Sanger direct sequencing, we detected mutations of these genes in tumor of 20 Glioblastma patients. We also found 5 somatic nonsynonymous coding mutations in 4 candidate genes, among which 2 mutations located in the PEST region of Mcl-1 (D155G and L174S). We then expanded the sample pool by sequencing Mcl-1 in another 43 GBM patients, finding another somatic mutation in the same region (D155H). At the same time we detected the expression of Mcl-1 at the mRNA and protein levels in tumor samples and normal tissue samples. It showed that somatic mutation could interfere the degradation of Mcl-1 protein. Compared with the relatively overexpressing wild-type Mcl-1 cells, glioma cells which been transfected with mutant genes grew much faster. Meanwhile, we found that nude mice injected of glioma cell with transfection mutations grew significantly weaker than the nude mice injected relative Mcl-1 in the nude mice experiments. The size of the tumor in the mutation nude was larger than that of the wild nude mice.In this study, we for the first time identified several mutations including Mcl-1, and identified the gene as a new therapeutic target of the treatment of GBM.Part I The identification of myeloid cell leukemia-1 (Mcl-1) mutations in GBM.Objective:(1) To investigate the distribution of gene mutation in normal.tissue and tumor tissue. (2) To analyse the relationship of Mcl-1 mutation and expression of Mcl-1. (3) To discuss the protein function after gene mutation.Methods:(1) Direct sequencing method was applied to detect 20 tumor-related genes in the tumor tissue and blood samples respectively in 20 patients. Additional 43 patients was detected to identify whether there was a corresponding location of the mutation. (3) Tumor tissues and corresponding normal tissuesMcl-1 protein levels change was detected by Western blot assay.Results:(1) By direct sequencing, we screened 20 tumor-related genes that were either well described in the literature or observed multiple times in human cancer sequencing efforts, in the cancerous samples and normal control from 20 histologically confirmed GBM patients. We also found 5 somatic nonsynonymous coding mutations in 4 candidate genes, among which 2 mutations located in the PEST region of Mcl-1 (D155G and L174S). We then expanded the sample pool by sequencing Mcl-1 in another 43 GBM patients, finding another somatic mutation in the same region (D155H). (2) By the results of immunohistochemistry, we found expression of Mcl-1 in the tumor tissue was obviously higher than that of normal tissue, while by RT-PCR experiments, we found that gene expression level of Mcl-1 in the tumor tissue is significantly higher than that in the normal tissue. (3) We got the same conclusion in Western blot.Conclusions:(1) We found a number of tumor-related mutations in tumor tissue, and for the first time, founding the gene mutation of Mcl-1. (2) Mcl-1 protein was significantly increased in gliomas, and might have a certain amount of changes in the relationship between the development of tumors. (3) We found Mcl-1 protein expression level in tumor tissue was significantly higher than that in the normal tissue, and it was statistically significant.Part Ⅱ The effects and possible mechanisms of Mel-1 mutation in the progressions of glioblastoma proliferation, apoptosis and migrationObjective:(1) To identify Mcl-l’s function on phosphorylation of AKT pathway. (2) To detect the half-life of Mcl-1 protein in both wild and mutant cells (3) To compare the Cell proliferations and apoptosises in wild Mcl-1 cells and mutant Mcl-1 cells. (4) To compare the migrations of wild Mcl-1 cells and mutant Mcl-1 cells; (5) To compare the tumor volume in nude mice.Methods:(1) Through the transient transfection transferring Mcl-1 gene into tumor cells and inducing cell direct mutatio. By detecting pathway proteins, we wanted to indentify the post-transcriptional process. (2) Half-life of Mcl-1 protein in wild cells and mutant cells were detected by 35S methionine pulse-chase label method. (3) The proliferation of wild cells and mutation cells were detected by MTT assay and the apoptosis of two groups were detected by flow cytometry. (4) The migration of wild Mcl-1 cells and mutant Mcl-1 cells were indentified by transwell experiment.Results:(1) We did not find the changes of phosphorylated Akt pathway proteins in wild cells and mutation cells. (2) Compared to non-mutant cells, mutant cells had significantly prolonged half-life of Mcl-1 protein. (3) The survival rate of the cells which were transfected with wild-type Mcl-1 was significantly lower compared with mutants in U251 cells treated with TRAIL.we found Mcl-1 mutants transfection suppressed TRAIL-induced apoptosis to a greater extent than wild-typein cells. (4) Compared to non-mutate cells, the migration ability of mutant cells enhanced obviously. We also found in the nude mice experiment that the tumor was bigger in mutant ones than that in the non-mutant mice.Conclusions:(1) phosphorylated Akt pathway was not changed in the mutant cells. (2) The half-life of Mcl-1 in mutant cells is prolonged. (3) The viability and anti-apoptotic effect of Mcl-1 mutation cells were enhanced. (4) Cell invasion and migration of Mcl-1 mutant cells were enhanced. |
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