内源性逆转录病毒基因Np9在人白血病细胞中的转录调控机制研究

The viral Np9 transcript, encoded by HERV-K type 1, has been shown to be exclusively present in tumor cells. Recently, we identified that the native Np9 protein was highly expressed in 56% of leukemia patients but rarely detected in normal blood cells. Moreover, the expression of Np9 significantly promoted the growth of human myeloid and lymphoblastic leukemia cells. However, the mechanism for activation of Np9 transcription in leukemia has hitherto vaguely understood. It was reported that the 5'LTR of HERV-K proviruse contained the promoter and enhancing elements. Besides, an early study noted the CpG methylation directly regulated transcriptional activity of HERV-K in melanoma cells. We recently obtained the sequences of HERV-K Np9 5'LTR from K562 cells which had high content of Np9 protein. Furthermore, we used demethylating agent 5-Aza-dC to treat K562 cells and found strong increase of Np9 mRNA by Real-time PCR. Based on these previous studies, we sought to indentify the core region of Np9 promoter, the specific transcription factors and reveal that how methylation regulate the binding of transcription factors on promoter, which shall provide new ideas for the correlation between HERV-K Np9 and leukemia pathogenesis.

HERV-K(人内源性逆转录病毒K)Np9基因在肿瘤细胞中呈特异性转录激活。新近，申请人所在团队在国际上率先证明Np9基因在白血病细胞中呈高转录激活状态。其编码的蛋白在56%的白血病患者中高表达，而在正常人样本中几乎不表达，并且该基因的激活能显著促进白血病细胞的增殖。然而，Np9基因在白血病细胞中是如何被转录激活迄今仍未明确。已知HERV-K前病毒5'LTR（长末端重复序列）被认为含有增强和起始转录的有效元件，并且在黑色素瘤细胞中，启动子通过去甲基化后被激活转录。申请人已从白血病细胞K562中获得Np9前病毒5'LTR，并用去甲基化试剂5-Aza-dC处理细胞，发现Np9的转录水平得到显著上调。因此，申请人拟在此基础上寻找白血病细胞中Np9启动子核心区域、鉴定关键转录因子、分析转录因子结合位点甲基化程度及揭示甲基化对启动子活性的影响，为探索白血病病毒病因学提供全新思路。

HERV-K(人内源性逆转录病毒K)Np9基因在肿瘤细胞中呈特异性转录激活其。编码的蛋白在56%的白血病患者中高表达，而在正常人样本中几乎不表达，并且该基因的激活能显著促进白血病细胞的增殖。HERV-K Np9前病毒5’LTR（长末端重复序列）被认为含有增强和起始转录的有效元件，本项目通过构建不同长度的5’LTR序列载体，利用双荧光素酶报告基因系统分析了转录核心区域；运用DNA Pull Down联合质谱技术获得了与转录序列相结合的蛋白。为深入研究该基因的转录调控奠定了基础。另一方面，本项目首次发现并证实Np9蛋白可以特异地和调控白血病干祖细胞更新和增殖的beta-catenin蛋白直接相互作用，显著增加beta-catenin蛋白的含量及核内分布；在白血病细胞中过表达Np9蛋白会引起染色体结构不稳定，对比低表达目的基因的细胞，多产生了48对融合基因；与此同时过表达Np9蛋白还会导致DNA损伤修复标志rH2AX的增高，这强烈提示我们Np9可能具有引起白血病细胞基因组不稳定的功能。以上结果具有一定的科学意义和潜在的临床价值，是具有原创性的研究。