EZH2基因调控脑胶质瘤细胞恶性生物学行为的实验研究
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摘要
脑胶质瘤是人类恶性程度最高和最难治愈的肿瘤之一。星形细胞瘤,少枝胶质细胞瘤以及混合性胶质瘤在成人原发性中枢神经系统肿瘤中所占比例超过80%。尽管采取了各种积极地治疗,据最新的流行病学调查,对于低级别的胶质瘤,问变性星形细胞瘤及胶质母细胞瘤,其2年生存率分别为66%,45%和9%。(目前,脑胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗和综合治疗等,尽管近年来上述治疗措施已取得长足进步,但由于胶质瘤呈浸润性生长,与周围脑组织无明显分界且易于复发,且疗效均不甚理想。新的治疗手段如基因治疗等虽有望成为治愈胶质瘤的途径之一,但是目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质瘤细胞恶性生物学行为的分子靶标,明确其在胶质瘤发病过程中的病理机制,已成为神经外科领域的研究热点之一
EZH2 (Enhancer of zeste homolog 2)是聚硫蛋白复合物基因家族(PcG)重要成员之一,在PcG基因家族中,EZH2起着核心作用。通过对核小体的修饰、染色体的重塑及干扰其他转录因子的转录调节,EZH2在胚胎的发育、细胞增殖及周期调节中起着重要作用。近年来,越来越多的关注转向到研究EZH2的表达模式。在一系列人类原发肿瘤中,运用mRNA原位杂交的方法发现,相对于正常组织,肿瘤中EZH2处于高表达状态。而且研究B细胞非何杰金氏淋巴瘤发现,与正常组织相比,在中、高级非何杰金氏淋巴瘤中,BMI-1和EZH2存在很高程度的共表达。Brachen等人证实PRB-E2F通路控制EZH2和EED的表达,EZH2和EED的转录调控需要E2F转录因子的参与。逐渐升高的EZH2表达可以缩短细胞周期中Gl期,从而有助于原发性MEFs的增殖。
目前,EZH2作为一种全新的癌基因在前列腺癌、乳腺癌、肾细胞癌和肝癌中的表达显著升高,而对于其在脑胶质瘤发生中所扮演的功能角色却鲜有报道。EZH2很有可能在胶质瘤形成的过程中发挥了重要作用。因此,探讨EZH2参与脑胶质瘤发生发展以及恶变的分子病理机制,有助于明确胶质瘤的恶性生物学行为及机理。
本研究目的在于通过检测EZH2在不同级别胶质瘤组织及胶质瘤细胞株中的表达模式,及对其在胶质瘤细胞株的表达予以干预敲减,以期阐明其在胶质瘤发生学中作用的具体机制。实验共分3部分：第一部分主要检测不同级别胶质瘤及正常脑组织中EZH2的表达水平;第二部分主要查明其在胶质瘤细胞株中的表达模式；最后在胶质瘤细胞株中利用siRNA敲减EZH2,了解其对胶质瘤增殖及凋亡的影响。
目的：研究EZH2基因在脑胶质瘤组织和正常脑组织中表达水平。
方法：根据EZH2的cDNA序列设计相应引物,收集37例不同级别脑星形胶质细胞瘤和8例正常脑组织,应用实时荧光定量PCR检测EZH2mRNA在上述组织标本中的相对表达水平,应用免疫组织化学染色检测EZH2在53例脑星形胶质细胞瘤组织和8例正常脑组织中的蛋白表达水平,并利用Spearman相关分析检验EZH2蛋白与增殖抗原Ki-67表达的相关性。最后应用免疫荧光／激光共聚焦技术获取EZH2蛋白的细胞定位信息。
结果：37例脑星形胶质细胞瘤和8例正常脑组织实时荧光定量PCR结果提示EZH2mRNA表达水平在星形胶质瘤组织中明显上调,与正常脑组织相比差别显著(176.96±122.69 vs 1.00±1.43；p=0.000)。并且,EZH2mRNA水平在高级别胶质瘤组织中的表达水平明显高于低级别胶质瘤,二者相比差异显著(248.33±76.25 vs28.27±29.69；p=0.000)。相似地,免疫组织化学染色提示EZH2蛋白在星形胶质细胞瘤组织中表达明显上调,与正常脑组织相比差别显著(12.10±8.45%vs0.0±0.0%；P=0.000),且高、低级别胶质瘤之间差别显著(16.67±6.89%vs3.91±2.98%；P=0.000),EZH2阳性细胞染色率与增殖抗原Ki-67蛋白表达呈明显的正相关关系(Spearman r=0.67, P<0.001)。免疫荧光／激光共聚焦提示EZH2蛋白定位于胶质瘤细胞核膜。
结论：EZH2表达于被检测的星形胶质细胞瘤和正常脑组织中,但其在星形胶质细胞瘤组织中的mRNA和蛋白表达水平明显高于正常脑组织,并和胶质瘤病理级别以及增殖抗原Ki-67表达呈正相关性,提示EZH2可能在星形胶质瘤的发生发展恶变过程中具有重要作用。
目的：研究EZH2在胶质瘤细胞系U251和U87MG中EZH2的mRNA.蛋白表达水平和细胞亚定位水平。
方法：应用实时荧光定量PCR等方法检测EZH2在胶质瘤细胞系U251和U87MG中的mRNA表达水平,应用蛋白印迹研究EZH2在胶质瘤细胞系中的蛋白表达水平,应用免疫荧光／激光共聚焦检测EZH2在胶质瘤细胞中的细胞亚定位水平及其与Ki-67蛋白的定位关系。
结果：荧光定量RT-PCR结果提示EZH2mRNA表达水平在胶质瘤细胞系U251和U87MG中明显上调,与正常脑组织相比差别显著(387.40±37.38,80.78±6.800 vs1.00±1.43；P=0.000)。免疫荧光／激光共聚焦提示EZH2蛋白主要表达于胶质瘤细胞胞核,并且部分表达EZH2细胞亦表达增殖抗原Ki-67,蛋白印迹实验证实其在U251和U87MG细胞系中的表达水平明显高于正常脑组织(0.64±0.01,0.36±0.004 vs 0.0±0.0;P<0.05)。
结论：EZH2在被检测的星形胶质瘤细胞系中,其mRNA和蛋白表达水平明显高于正常脑组织,EZH2在被检胶质瘤细胞株中蛋白定位在细胞核,与增殖抗原Ki67存在共定位现象。
目的：研究靶向EZH2的RNA干扰对U251胶质瘤细胞株增殖、凋亡的作用。
方法：设计并合成靶向EZH2的siRNA,脂质体法瞬时转染U251细胞株,优化转染条件。CCK-8法测定转染后24、48小时U251细胞增殖率,TUNEL测定干扰后24、48小时早期凋亡细胞比例和细胞周期改变,FACS检测EZH2siRNA48h后凋亡细胞比例,蛋白印迹实验测定转染后48小时Cyclin A2, Cyclin D1, Cyclin E1, C-MYB,B-MYB,E2F1, H3K27me3, H3K27me2, P53等蛋白表达水平变化。
结果：EZH2 siRNA可有效敲减U251细胞株EZH2的表达。CCK-8法检测EZH2-siRNA转染组0,24,48小时的细胞生长吸光值分别为(0.161±0.046,0.195±0.043,0.279±0.038)；较阴性对照组(0.154±0.037,0.346±0.041,0.599±0.033)和未转染组(0.167±0.043,0.365±0.055,0.672±0.039)降低;其中24、48小时细胞生长与对照组相比,差异均有统计学意义(P<0.05)。经流式细胞仪检测,24,48小时靶向siRNA转染引起U251胶质瘤细胞的早期凋亡率分别为(13.01±2.65%,16.24±2.14%),阴性对照组为(4.86±1.87%,5.82±1.35%),未转染组为(2.85±1.58%,3.13±1.73%)。在24、48小时,早期凋亡比例均高于对照组和未转染组,两者之间差异显著(P<0.05)。靶向EZH2siRNA转染U251细胞48小时后G1期细胞比例显著升高,较阴性对照组和未转染组差异显著(82.32±2.11%vs 39.37±2.33%,36.75±1.68%；P<0.05)。敲减EZH2的表达后,Cyclin A2, Cyclin D1, Cyclin E1, C-MYB,B-MYB,E2F1, H3K27me3的蛋白表达在干扰后24至48小时出现降低,与对照组相比差异显著；P53的表达在干扰后出现升高,与对照组相比差异显著。
结论：RNA干扰敲减EZH2在U251胶质瘤细胞中的表达可通过PRB-E2F、P53和H3K27me3甲基化途径,有效抑制肿瘤细胞的增殖和促进凋亡。有效敲减EZH2在胶质瘤中的表达有可能作为治疗胶质瘤的潜在策略。
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 tumors
Objective: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 lines
Objective: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 vitro
Objective: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.
EZH2 (Enhancer of zeste homolog 2)是聚硫蛋白复合物基因家族(PcG)重要成员之一,在PcG基因家族中,EZH2起着核心作用。通过对核小体的修饰、染色体的重塑及干扰其他转录因子的转录调节,EZH2在胚胎的发育、细胞增殖及周期调节中起着重要作用。近年来,越来越多的关注转向到研究EZH2的表达模式。在一系列人类原发肿瘤中,运用mRNA原位杂交的方法发现,相对于正常组织,肿瘤中EZH2处于高表达状态。而且研究B细胞非何杰金氏淋巴瘤发现,与正常组织相比,在中、高级非何杰金氏淋巴瘤中,BMI-1和EZH2存在很高程度的共表达。Brachen等人证实PRB-E2F通路控制EZH2和EED的表达,EZH2和EED的转录调控需要E2F转录因子的参与。逐渐升高的EZH2表达可以缩短细胞周期中Gl期,从而有助于原发性MEFs的增殖。
目前,EZH2作为一种全新的癌基因在前列腺癌、乳腺癌、肾细胞癌和肝癌中的表达显著升高,而对于其在脑胶质瘤发生中所扮演的功能角色却鲜有报道。EZH2很有可能在胶质瘤形成的过程中发挥了重要作用。因此,探讨EZH2参与脑胶质瘤发生发展以及恶变的分子病理机制,有助于明确胶质瘤的恶性生物学行为及机理。
本研究目的在于通过检测EZH2在不同级别胶质瘤组织及胶质瘤细胞株中的表达模式,及对其在胶质瘤细胞株的表达予以干预敲减,以期阐明其在胶质瘤发生学中作用的具体机制。实验共分3部分：第一部分主要检测不同级别胶质瘤及正常脑组织中EZH2的表达水平;第二部分主要查明其在胶质瘤细胞株中的表达模式；最后在胶质瘤细胞株中利用siRNA敲减EZH2,了解其对胶质瘤增殖及凋亡的影响。
目的：研究EZH2基因在脑胶质瘤组织和正常脑组织中表达水平。
方法：根据EZH2的cDNA序列设计相应引物,收集37例不同级别脑星形胶质细胞瘤和8例正常脑组织,应用实时荧光定量PCR检测EZH2mRNA在上述组织标本中的相对表达水平,应用免疫组织化学染色检测EZH2在53例脑星形胶质细胞瘤组织和8例正常脑组织中的蛋白表达水平,并利用Spearman相关分析检验EZH2蛋白与增殖抗原Ki-67表达的相关性。最后应用免疫荧光／激光共聚焦技术获取EZH2蛋白的细胞定位信息。
结果：37例脑星形胶质细胞瘤和8例正常脑组织实时荧光定量PCR结果提示EZH2mRNA表达水平在星形胶质瘤组织中明显上调,与正常脑组织相比差别显著(176.96±122.69 vs 1.00±1.43；p=0.000)。并且,EZH2mRNA水平在高级别胶质瘤组织中的表达水平明显高于低级别胶质瘤,二者相比差异显著(248.33±76.25 vs28.27±29.69；p=0.000)。相似地,免疫组织化学染色提示EZH2蛋白在星形胶质细胞瘤组织中表达明显上调,与正常脑组织相比差别显著(12.10±8.45%vs0.0±0.0%；P=0.000),且高、低级别胶质瘤之间差别显著(16.67±6.89%vs3.91±2.98%；P=0.000),EZH2阳性细胞染色率与增殖抗原Ki-67蛋白表达呈明显的正相关关系(Spearman r=0.67, P<0.001)。免疫荧光／激光共聚焦提示EZH2蛋白定位于胶质瘤细胞核膜。
结论：EZH2表达于被检测的星形胶质细胞瘤和正常脑组织中,但其在星形胶质细胞瘤组织中的mRNA和蛋白表达水平明显高于正常脑组织,并和胶质瘤病理级别以及增殖抗原Ki-67表达呈正相关性,提示EZH2可能在星形胶质瘤的发生发展恶变过程中具有重要作用。
目的：研究EZH2在胶质瘤细胞系U251和U87MG中EZH2的mRNA.蛋白表达水平和细胞亚定位水平。
方法：应用实时荧光定量PCR等方法检测EZH2在胶质瘤细胞系U251和U87MG中的mRNA表达水平,应用蛋白印迹研究EZH2在胶质瘤细胞系中的蛋白表达水平,应用免疫荧光／激光共聚焦检测EZH2在胶质瘤细胞中的细胞亚定位水平及其与Ki-67蛋白的定位关系。
结果：荧光定量RT-PCR结果提示EZH2mRNA表达水平在胶质瘤细胞系U251和U87MG中明显上调,与正常脑组织相比差别显著(387.40±37.38,80.78±6.800 vs1.00±1.43；P=0.000)。免疫荧光／激光共聚焦提示EZH2蛋白主要表达于胶质瘤细胞胞核,并且部分表达EZH2细胞亦表达增殖抗原Ki-67,蛋白印迹实验证实其在U251和U87MG细胞系中的表达水平明显高于正常脑组织(0.64±0.01,0.36±0.004 vs 0.0±0.0;P<0.05)。
结论：EZH2在被检测的星形胶质瘤细胞系中,其mRNA和蛋白表达水平明显高于正常脑组织,EZH2在被检胶质瘤细胞株中蛋白定位在细胞核,与增殖抗原Ki67存在共定位现象。
目的：研究靶向EZH2的RNA干扰对U251胶质瘤细胞株增殖、凋亡的作用。
方法：设计并合成靶向EZH2的siRNA,脂质体法瞬时转染U251细胞株,优化转染条件。CCK-8法测定转染后24、48小时U251细胞增殖率,TUNEL测定干扰后24、48小时早期凋亡细胞比例和细胞周期改变,FACS检测EZH2siRNA48h后凋亡细胞比例,蛋白印迹实验测定转染后48小时Cyclin A2, Cyclin D1, Cyclin E1, C-MYB,B-MYB,E2F1, H3K27me3, H3K27me2, P53等蛋白表达水平变化。
结果：EZH2 siRNA可有效敲减U251细胞株EZH2的表达。CCK-8法检测EZH2-siRNA转染组0,24,48小时的细胞生长吸光值分别为(0.161±0.046,0.195±0.043,0.279±0.038)；较阴性对照组(0.154±0.037,0.346±0.041,0.599±0.033)和未转染组(0.167±0.043,0.365±0.055,0.672±0.039)降低;其中24、48小时细胞生长与对照组相比,差异均有统计学意义(P<0.05)。经流式细胞仪检测,24,48小时靶向siRNA转染引起U251胶质瘤细胞的早期凋亡率分别为(13.01±2.65%,16.24±2.14%),阴性对照组为(4.86±1.87%,5.82±1.35%),未转染组为(2.85±1.58%,3.13±1.73%)。在24、48小时,早期凋亡比例均高于对照组和未转染组,两者之间差异显著(P<0.05)。靶向EZH2siRNA转染U251细胞48小时后G1期细胞比例显著升高,较阴性对照组和未转染组差异显著(82.32±2.11%vs 39.37±2.33%,36.75±1.68%；P<0.05)。敲减EZH2的表达后,Cyclin A2, Cyclin D1, Cyclin E1, C-MYB,B-MYB,E2F1, H3K27me3的蛋白表达在干扰后24至48小时出现降低,与对照组相比差异显著；P53的表达在干扰后出现升高,与对照组相比差异显著。
结论：RNA干扰敲减EZH2在U251胶质瘤细胞中的表达可通过PRB-E2F、P53和H3K27me3甲基化途径,有效抑制肿瘤细胞的增殖和促进凋亡。有效敲减EZH2在胶质瘤中的表达有可能作为治疗胶质瘤的潜在策略。
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 tumors
Objective: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 lines
Objective: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 vitro
Objective: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.
引文
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