| Gliomas are among the most aggressive and treatment refractory of all human tumors.Astrocytomas,oligodendrogliomas,and mixed tumors comprised over 80% of primary adult central nervous system tumors.With aggressive treatment,the 2-year survivals are 66,45,and 9%,respectively,for low-grade astrocytomas,anaplastic astrocytomas,and glioblastoma multiforme,according to the most recent Surveillance,Epidemiology,and End Result data.Current therapeutic modalities including surgical resections, chemotherapy, radiotherapy or combinations can not ensure a cure and, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytoma tumorigenesis may offer important targets for treatment strategies.The enhancer of zeste homolog 2(EZH2) is a key member of the polycomb group of genes(PcG),which is important for embryonic development,cellular proliferation and cell cycle regulation through nucleosome modification,chromatin remodeling,and interaction with other transcription factors. Over the past few years,increased attention has been given to EZH2 expression pattern.A series of primary human tumors were tested for EZH2 expression through mRNA in situ hybridization highlighting that EZH2 is highly expressed in tumors versus normal tissue.Moreover,a variety of B-cell non-Hodgkin lymphomas have been extensively analyzed for BMI-1 and EZH2,while their expression is mutually excluded in normal tissue. Brachen et al provided a conclusion that PRB-E2F pathway controls the expression of EZH2 and EED and that the E2F transcription factors are required for the transcriptional regulation of EZH2 and EED.Ectopic expression of EZH2 shortens the G1-phase of the cell cycle and confer a proliferative advantage in primary MEFs.Currently, as a bona fide oncogene, EZH2 is significantly overexpressed in a variety of malignancies including breast, prostate cancer, renal cell carcinoma, and hepatocellular carcinomas, but few investigations about the the functional role of EZH2 in the carcinogenesis of glioma cells have been repored. EZZH2 might play an important role in the malignant transformation of tumor cells. Hence, further investigation of the functional role of EZH2in the carcinogenesis of glioma cells may offer a better understanding of malignant behavior of glioma cells.The aim of this study is to clarify the exact role for EZH2 in the oncogenic process of glioma cells by examining its expression pattern in glioma tissues of different grades and glioma cell lines and knocking down its expression level in glioma cell lines. This study is consisted of three main parts:the first part is to investigate the expression level of EZH2 gene in astrocytomas of different grades and normal brain tissues, the second is to investigate the expression pattern of EZH2 in glioma cell lines and finally, the effects of siRNA targeting human EZH2 on tumor proliferation and apoptosis are evaluated in U251 glioma cells.Part I Expression of the enhancer of zeste homolog 2(EZH2) in astrocytic tumorsObjective:To investigate the expression level of EZH2 gene in astrocytomas of different grades and normal brain tissues.Methods:The expression levels of EZH2 mRNA were evaluated by real-time quantitative PCR in 37 astrocytic tumors and 8 normal brain tissue samples. By using immunohistochemistry, expression levels of EZH2 protein were assessed in 53 astrocytic tumors of different pathological grades and 8 normal brain controls. Further, the correlation between EZH2 and Ki-67 immunoreactivity was examined with the Spearman's correlation coefficient. Finally, confocal and fluorescence microscopy was employed to investigate the celluar location of EZH2 protein in astrocytomas.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of EZH2/β-actin in high-grade astrocytomas versus low-grade (248.33±76.25 vs 28.27±29.69,respectively; p=0.000) or normal brain tissues (176.96±122.69 vs 1.00±1.43, respectively; p=0.000). Further, EZH2 immunoreactivity was predominantly detected in the cytoplasm of tumor cells, whereas no positive staining for EZH2 was observed in normal brain tissues. Statistical analysis showed increased EZH2 labelling index in high-grade astrocytomas versus low-grade tumors 16.67±6.89% vs 3.91±2.98%, respectively; P=0.000) or normal controls (12.10±8.45% vs 0.0±0.0%, respectively; P=0.000). Importantly, EZH2 staining positively correlated with the proliferative activity indicated by Ki-67 staining (P<0.001, r=0.67, by Spearman's correlation coefficient). Confocal and fluorescence microscopy assay demonstrated the localization of EZH2 around the nuclear membrane of glioma cells.Conclusion:In summary, we demonstrate that EZH2 is present in either astrocytomas or normal brain tissues examined, and its expression positively correlates with the degree of malignancy at both RNA and protein levels. These data suggest that overexpression of EZH2 may serve as one important molecular mechanism that underlies the development and progression of astrocytic tumors.Part II Expression of the enhancer of zeste homolog 2(EZH2) in U251 and U87MG glioma cell linesObjective:To investigate the expression pattern of EZH2 and investigate the celluar location of EZH2 protein in glioma cell lines.Methods:The expression level of EZH2 mRNA was also evaluated by real-time quantitative PCR in these two glioma cell lines. Confocal and fluorescence microscopy assay was employed to investigate the celluar location of EZH2 protein in U251 and U87MG glioma cell lines.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of EZH2/β-actin in U251 and U87MG glioma cells versus normal brain tissues (387.40±37.38,80.78±6.800 vs 1.00±1.43, P=0.000). EZH2 immunoreactivity was predominantly detected in the nucleus of tumor cells by Confocal and fluorescence microscopy assay. Western blot analysis showed increased EZH2 expression levels in glioma cells versus normal brain tissues (0.64±0.01,0.36±0.004vs 0.0±0.0; P<0.05). Confocal and fluorescence microscopy assay demonstrated a close co-localization of EZH2 and Ki-67 in the nucleus of glioma cells.Conclusion:EZH2 is present in glioma cell lines examined, and its expression levels significantly increase at both mRNA and protein levels versus normal brain tissues. A close co-localization of EZH2 and Ki-67 in the nucleus of glioma cells was demonstrated.PartⅢRNA interference targeting EZH2 inhibits glioma cell proliferation, enhance cell apotosis in vitroObjective:To investigate the effects of siRNA targeting human EZH2 on tumor proliferation and apoptosis of U251 glioma cells.Methods:The siRNA targeting human EZH2 were transfected into U251 glioma cells. Cell proliferative activity was assessed by CCK-8 assay and, cell apoptosis was analyzed by flow cytometry assay. The expression levels of yclin A2, Cyclin D1, Cyclin E1, C-MYB,B-MYB,E2F1, H3K27me3, H3K27me2 and P53 Protein were measured by western blot.Results:The expression level of EZH2 protein was knocked down by EZH2 siRNA as indicated by western blotting analysis. Proliferation of U251 glioma cells was significantly reduced compared with that of control at 24,48h post transfection.The flow cytometric analysis showed increased proportion of annexin V positive cells in EZH2 siRNA group at 24 and 48h post transfection versus si-scrambled and untransfected groups(13.01±2.65% vs 4.86±1.87%,2.85±1.58% at 24h; 16.24±2.14% vs5.82±1.35%,3.13±1.73% at 48 h post transfection).Expression levels of Cyclin A2, Cyclin Dl, Cyclin El C-MYB,B-MYB,E2F1 and H3K27me3 proteins siginificantly decreased at 24 and 48h after knocking down EZH2 expression. Expression levels of P53 increased significantly post transfection of EZH2 siRNA.Conclusion:Overall, our results indicate that siRNA transfection targeting EZH2 can inhibit cell proliferation and induce apoptosis through a P53 dependant way. Given the strong upregulation of EZH2 expression in malignant gliomas, knock-down of EZH2 expression may therefore represent a potentially treatment strategy against glioma.
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