| Glioma is common and owning a high inciddence rate in population,which occupies the first place in the malignant tumor in the nervous system.As a dangerous killer to human health,the glioma is closely related to ionizing radiation and genetic susceptibility.However,the exact etiology of glioma is remain unclear.Radiation therapy is one of the most important treatments for malignant tumors,and currently the standard treatment of glioma is mainly combined with radiotherapy and chemotherapy.Nevertheless,the high grade glioma(HGG)still own high recurrence rates,high risk of metastasis and low survival rates.The main reason for the treatment failure is the remarkable invasion of the HGG cells,which show significant resistance to radiation therapy,and possiblely existing tumor stem cells involved in this process.The glioma stem cells have high cloning activity,aggressive and resistant to traditional chemotherapy and radiotherapy.The zeste gene enhancer homolog(enhancer of zeste homolog2,EZH2)and phosphoinositide 3-kinase(PI3K)/AKT signaling pathway were found expressed in a variety of tumors.Some studies showed that the increased expression of EZH2 was detected in the glioma tissue and was associated with a poor survival prognosis in patients.However,there are few related molecular mechanisms explored for the reason of the adverse prognosis of EZH2,especially the radiobiologic effects of EZH2 expression after radiation therapy.This study mainly focus on the the expression and clinical significance of PI3K,phosphorylated AKT(P-AKT)and EZH2 in glioma tissues,as well as the radiation resistance caused by the signal pathway axis in the glioma cells,to explore their biological function and molecular mechanisms in vitro.Part I A clinical analysis of 67 cases of postoperative high grade glioma treated with radiotherapy combined with temozolomideTo retrospectively assess the efficacy and side effects of radiotherapy combined with temozolomide(TMZ)for patients with high grade glioma,and to explore the clinical prognostic factors,67 patients diagnosed with high-grade glioma who were received treatment from June 1,2009 to February 29,2016 were selected,and the treatment response and side effects were evaluated based on clinical characteristics.The survival rate was calculated by Kaplan-Meier method and analyzed by the Log-rank test using software SPSS(version 20.0),and Cox regression model was used for multivariate analysis.The results showed that the 2-,3-year overall survival(OS)rates were 28.4%and 6.0%,respectively;and the corresponding progression-free survival(PFS)rates were 20.1%,and 4.5%.Univariate analysis showed that pathological grade(χ2=6.95,p<0.05),extent of resection(χ2=10.8,p<0.05)were important factors affecting the survival rates of patients,meanwhile age(χ2=5.9,p<0.05),the extent of resection(χ2=10.57,p<0.05)and the cycles of TMZ chemotherapy(χ2=4.64,p<0.05)were prognostic factors for PFS.Multivariate regression analysis showed that pathological grade and extent of resection were independent prognostic factors for OS in HGG.Nevertheless,age,extent of resection and the cycles of TMZ chemotherapy affected PFS independently.Few patient experienced severe adverse effects such as hematologic toxicity,liver dysfunction and gastrointestinal toxicity.Conclusions The treatment modality of 3D-CRT and IMRT combined with TMZ failed to result in a longterm survival for HGG patients.Pathological grade and extent of resection are independent prognostic factors for patients with HGG.Part Ⅱ Clinical analysis of EZH2,PI3K and P-AKT expression in glioma tissuesThe clinical and pathological data of 74 patients with glioma were collected,and the survival rate,the recurernce rate and metastasis of the patients were preliminarily analyzed.The expression of EZH2,PI3K,P-AKT protein in glioma tissues were detected by immunohistochemical staining,and 12 fresh glioma specimens and normal brain tissues were obtained and tested by Western blot method.The corelation between the expression level of proteins in glioma and the prognosis of clinical survival were analyzed.The results showed that the pathological grade,complete resection,KPS score,were the independent prognostic factors.Higher expression of EZH2 or P-AKT were adverse factors in the patients with a lower survival rate,and the 3 year OS rates were 9.1%and 17.0%,respectively.The rate of the expression of EZH2 and P-AKT in glioma tissues was obviously enhanced accompany with the rise of WHO pathological grading.Further statistical analysis showed that the expression level of EZH2,PI3K and P-AKT proteins were positively correlated,having a coexpression synergistic effect.EZH2 in the grade IV of high grade glioma was significantly higher than that in grade Ⅱ.The survival benefit was obtained in the group with low co-expression level of EZH2 and P-AKT,and OS was significantly higher than that in the high expression group(p<0.05).Interestingly,The most obvious group is the combination of co-expression of the three poteins mentioned above.The total high co-expression group had a better significant survial benefit when compared with the whole lowco-expression group(p<0.001),which provided new molecular prognostic indicators for the risk of clinical evaluation and treatment.The level of the expression of EZH2 and P-AKT is significantly correlated with the pathological grade.As prognostic indicators of glioma,it can serve as some certain clinical factors for evaluation of the recurrence and survival in glioma.Part Ⅲ Study on irradiation Resistance and Molecular Mechanism of PI3K/AKT and EZH2 in Glioma cellsThe expression level of EZH2,PI3K and P-AKT protein at different time points after irradiation in glioma cells was detected using the Western blot method in vitro experiments,and the higher expression of the biomarkers protein SOX2,Ecad and FOXA2 in glioma stem cells was further confirmed by the antibody chip experiment.Immunofluorescence assay was used to detect the dynamic changes of protein expression levels of EZH2,P-AKT and SOX2 in glioma cells at different time points after irradiation.In order to explore the molecular factors that causes irradiation resistance,the mRNA gene expression chip was used to screen the related genes.The changed molecular factors after radiation in glioma cells were mediated by PI3K,AKT and EZH2 signal axis,and the differentially expressed genes were tested.The results showed that the expression of EZH2,PI3K,and P-AKT protein in glioma cells after irradiation were significantly increased.The immunofluorescence test demostrated that the expression of EZH2,PI3K,P-AKT and SOX2 in glioma cell lines increased obviously at different time points after irradiation,and showing a linear increasing trend.Gene chips study indicated that,significant changes in mRNA expression level in EZH2,PI3K,and AKT were detected before and after the irradiation.The tumor stem cell biomarkers ALDH1A1 and SOX2 genes were detected increasing significantly in glioma stem cells,which preliminarily confirmed the hypothesis of radiation resistance caused by tumor stem cells.The activated NF-κB signaling pathway,arrested cell cycle,damaged double-strand DNA repair,activated oncogene,inhibition of apoptosis and epithelial-mesenchymal transition are all the molecular factors caused irradiation resistance in glioma cells.In vitro study indicated that the morphological changes of glioma cells at different time intervals after radiation.The expression levels of PI3K,AKT and EZH2 proteins were increased after irradiation of glioma cells.The expression level of stem cell related markers was analyzed in glioma cells after irradiation.Gene chips study indicated that,significant changes in mRNA expression level in EZH2,PI3K,and AKT were detected before and after the irradiation.The tumor stem cell biomarkers ALDH1A1 and SOX2 genes were detected increasing significantly in glioma stem cells,which preliminarily confirmed the hypothesis of radiation resistance caused by tumor stem cells.The activated NF-κB signaling pathway,arrested cell cycle,damaged double-strand DNA repair,activated oncogene,inhibition of apoptosis and epithelial-mesenchymal transition,all the molecular factors contributed to irradiation resistance in glioma cells. |
PDF Full Text Request