鼻咽癌中Myocardin转录失活机制及其功能研究

【作者】 陈浮；

【导师】 王胜资； 王纾宜； 邹丽芬；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 心肌素(Myocardin)基因是2001年在小鼠心脏cDNA文库中分离克隆的。因其在成年小鼠心脏的特异表达及其调控心肌分化重要作用而命名。它属于SAP结构域核蛋白家族并能与血清效应因子(serum response factor, SRF)结合,刺激心肌启动子。它在心肌和平滑肌的生长分化中是不可或缺的。研究表明,它参与调节缺氧导致的肺血管重塑、阿尔茨海默病中动脉过强收缩及脑血流下调、心肌肥大、扩张性心肌病的发病等。而新近的研究表明,心肌素在一些肿瘤中起负生长调节作用,可能是肿瘤抑制基因。本次我们研究了鼻咽癌中Myocardin基因转录失活机制及其在鼻咽癌中的作用。首先,我们检测了5个鼻咽癌细胞系Myocardin转录表达水平,发现3个表达沉默,1个表达下调。提示Myocardin转录失活可能在NPC中是一个普遍现象,并参与癌变过程。然后,我们用MSP方法检测了鼻咽癌细胞株、肿瘤组织、正常鼻咽上皮细胞株及正常上皮组织的Myocardin甲基化状态,结果显示4/5的鼻咽癌细胞株中Myocardin甲基化,鼻咽癌组73.8%的病例可检测到甲基化,而正常鼻咽组织则全部为非甲基化,差异具有显著性(p<0.01),说明NPC中Myocardin的甲基化具有肿瘤特异性。再次,我们用甲基化转移酶抑制剂对4个鼻咽癌细胞系进行处理,发现其能显著恢复Myocardin的表达,提示启动子甲基化是Myocardin表达下调的直接原因。最后,为明确Myocardin在NPC中的作用,我们利用Myocardin真核表达载体转染鼻咽癌细胞获得了稳定转染Myocardin的细胞株。功能试验结果显示,Myocardin可抑制鼻咽癌细胞生长、降低克隆形成效率。结果说明Myocardin在NPC中因为启动子CpG岛甲基化而失活,Myocardin可抑制鼻咽癌细胞的恶性生物学行为,是NPC相关的潜在肿瘤抑制基因。更多还原

【Abstract】 Background:Nasopharyngeal carcinoma (NPC) is an serious health problem in southern China and south-eastern Asia while the disease is very rare in most parts of the world. The tumorgenesis of NPC is proposed to be a multistep process; besides its significant association with Epstein-Barr Virus infection, epigenetic inactivations of tumor suppressor genes (TSG) and other genes play indispensable roles in its carcinogenesis. Exploring novel genes that are targets of hypermethylation in NPC may provide further insights into the development of this unique malignancy and open the way to discover novel diagnostic and therapeutic strategies. To identify novel candidate TSGs in NPC, we performed a genome-wide screening for genes inactivated by promoter hypermethylation. By analyzing changes in global gene expression profiles in two NPC cell lines before and after treatment with the combine treatment of the a demethylating agent 5-aza-2O-deoxycytidine (5-aza-dC) and the histone deacetylase (HDAC) inhibitor TSA, we identified Myocardin was downregulated by promoter hypermethylation in NPC.Methods:Transcriptional expression levels of Myocardin in NPC cells and normal nasopharyngeal epithelia were evaluated by semiquantitave RT-PCR. Methylation status of Myocardin in NPC cells, primary tumors and normal nasopharyngeal epithelia were addressed by methylation specific PCR and bisulfate genomic sequencing. The complete CDS of Myocardin gene were coloned into the expression vector pCMV-3Tag3A and transfected into CNE2 cells. Stable transfectance of Myocardin were selected by geneticin. The properties of Myocardin as a tumor suppressor gene were address by cell proliferation assay and colony formation assay.Results: Myocardin mRNA expression were inactivated in four out of five NPC cell lines. Myocardin were aberrantly methylated in 80%(4/5) NPC cell lines and 73.8%(48/65) of NPC primary tumors, but not in any of the twelve normal nasopharyngeal tissues. Loss of Myocardin expression can be completely restored by the methyltransferase inhibitor 5-aza-dC in four NPC cell lines. Ectopic expression of Myocardin in the Myocardin-silenced and NPC cell line CNE2 shows that Myocardin could inhibit cell cycle progression, colony formation.Conclusions:Epigenetic inactivation of Myocardin is frequent and tumor specific event in NPC. Myocardin might be considering as a candidate tumor suppressor gene in NPC.更多还原