EB病毒ncRNA在Burkitt淋巴瘤发病中的作用及机制研究

A central role for host-cellular ncRNA in the establishment and progression of human tumors has begun to emerge. Recent reports indicate that viral ncRNA can manipulate host-cell gene expression via the miRNA pathway and enhances its oncogenic property.Our findings also suggest that EBV encodes mir-BART5 to promote host cell canceration. Many papers report on the expression profiling of viral ncRNA in a wide variety of infected normal and neoplastic tissues that show there are distinct patterns of miRNA expression associated with different infection stage. We propose that the expression profiling of EBV ncRNAs in a wide variety of infected normal and neoplastic cells were tested by microarray analysis. Overexpression and lossof-function experiments will be performed to characterize the function of distinct expression EBV ncRNAs. EBV ncRNAs expression vectors will be made by inserting ncRNAs into eukaryotic expression vector and transfected into cells. The tumorigenicity of the transfected cells will be determined by the capacity to form colonies in agar and tumors in nude mice.The procedure will also involve decreasing expression of a EBV ncRNA by transfecting a coding gene for a shRNA that initiates degradation of the RNA by an RNAi. The interreaction of EBV ncRNA with host-cellular miRNA or mRNA will be tested by Northern blot and co-immunoprecipitation.We expect to reveal a new molecular mechanism through which EBV ncRNAs confers resistance to apoptosis in cells. The EBV ncRNAs might represent new therapeutic targets for the treatment of Burkitt's lymphoma.This study could also represent a novel approach to aid in the discovery of the role that EBV plays in oncogenesis.

内源性ncRNA表达紊乱与肿瘤发生相关；近期发现外源性（病毒）ncRNA可调控宿主细胞生理活动，可能在细胞癌变中起重要作用。我们也发现在细胞中高表达EBV miR-BART5能促进细胞恶性表征。EBV感染阳性正常细胞和淋巴瘤细胞之间，病毒ncRNA表达存在差异，提示不同的病毒ncRNA在各感染阶段中作用不同。本研究以EBV感染密切相关的Burkitt淋巴瘤为研究对象，从EBV感染阳性的正常细胞和肿瘤细胞中筛选出差异表达的病毒ncRNA；构建表达病毒ncRNA 的细胞株进行体外软琼脂集落生长和裸鼠移植瘤实验，并运用RNAi实验反向验证；通过免疫共沉淀和Northern blot 检测病毒ncRNA与宿主miRNA或mRNA的相互作用，阐述其致癌作用。期望发现EBV ncRNA在肿瘤形成机制中的新线索，为临床提供Burkitt淋巴瘤的诊治的新靶点，亦为研究病毒致癌机制提供新思路和新方法。

内源性ncRNA表达紊乱与肿瘤发生相关；近期发现外源性（病毒）ncRNA可调控宿主细胞生理活动，可能在细胞癌变中起重要作用。我们也发现在细胞中高表达EBV miR-BART5能促进细胞恶性表征。EBV感染阳性正常细胞和淋巴瘤细胞之间，病毒ncRNA表达存在差异，提示不同的病毒ncRNA在各感染阶段中作用不同。本研究以EBV感染密切相关的Burkitt淋巴瘤为研究对象，从EBV感染阳性的正常细胞和肿瘤细胞中筛选出差异表达的病毒ncRNA；构建表达病毒ncRNA 的细胞株进行体外软琼脂集落生长，并运用RNAi实验反向验证；通过免疫共沉淀和Northern blot 检测病毒ncRNA与宿主miRNA或mRNA的相互作用，阐述其致癌作用。期望发现EBV ncRNA在肿瘤形成机制中的新线索，为临床提供Burkitt淋巴瘤的诊治的新靶点，亦为研究病毒致癌机制提供新思路和新方法。.生物信息学分析显示mir-BART-5与 miR-18序列相近，miR-18 的靶基因表达的结缔组织生长因子（CTGF ）可促进细胞增殖。在鼠成纤维细胞 NIH/3T3 中瞬时转染 mir-BART-5-mimics 使其表达上调，采用Western blot 检测对照组与转染组细胞的CTGF 表达，采用体外软琼脂集落生成试验观察两组细胞生长的恶性表征。 转染组细胞在软琼脂上呈恶性表征.. 构建了 EBV B淋巴细胞与其Raji淋巴瘤细胞miRNA差异基因表达谱，筛选出115个差异表达基因，包括 45 个上调基因和 70 个下调基因,结果表明 EBV 感染淋巴瘤发生是一个涉及多个通路、多种基因，且病毒基因与宿主基因相互调控的复杂过程。了解EB病毒感染与鼻咽癌患者血清中相关抗体水平的关系,VCA/IgA、EA/IgA和Rta/IgG三种抗体的联合血清学检测，可较全面的反映EB病毒所处的感染时期，有极好的互补作用，有利于提高海口地区鼻咽癌的诊断率