去甲基化增强OY-TES-1表达及特异性CTL杀伤肝癌细胞的研究

Our previous study found that OY-TES-1 specifically expressed in liver cancer tissue，but its expression presented heterogenesis. Based on OY-TES-1 gen enriched CpG sits, the relation of expression and methylation can be speculated. Therefore, Following contents will be studied: (1) methylating regulation of OY-TES-1 expression. The methylated status of OY-TES-1 will be tested by SP and Pyrosequence, and its clinical significance will be analyzed. To find out the key sites of methylated reguation the reporter gene activity will be observed by cells transfected with luciferase reporter plasmid and treated with demethylated drug. (2) The methylated intervention affects the role of killing tumor cells by OY-TES-1 specific cytotoxic T lymphoycyte (CTL). With DC transfected with OY-TES-1 expression vector or and incubated with recombinant protein specific CTL was induced in vitro and observed it killing ability. This project will interpret the regulating mechanism of OY-TES-1 expression from epigenetic aspect and provide experimental data for treatment of liver cancer by methylating intervention.

我们前期研究发现，OY-TES-1在肝癌组织特异性表达,但存在表达异质性；鉴于OY-TES-1基因富含CpG位点,推测其表达与甲基化有关。故拟开展以下研究:（1）甲基化对OY-TES-1表达的调控：用BSP和焦磷酸测序分析肝癌组织OY-TES-1甲基化状态及临床意义；用OY-TES-1启动子报告基因载体转染细胞,观察去甲基化药物对启动子活性的影响,探寻甲基化调节的关键位点；借助ChIP和EMSA实验了解甲基化对转录因子Sp1与OY-TES-1启动子结合的影响；（2）甲基化干预对OY-TES-1特异性CTL杀瘤效应的影响：用OY-TES-1表达载体转染或/和重组蛋白孵育DC,体外诱导特异性CTL,用去甲基化药物诱导肝癌细胞表达OY-TES-1和HLA-I,观察CTL杀伤力。本项目从表观遗传学角度阐明OY-TES-1的表达调控机制,并为将甲基化干预用于OY-TES-1肝癌免疫治疗提供实验依据。

本研究主要结果如下：（1）Sequenom MassARRAY法检测肝癌组织OY-TES-1启动子区24个CpG位点的甲基化水平，结果显示：OY-TES-1启动子区总甲基化水平约20%，各CpG位点甲基化水平为7%~29%之间；进一步结合临床参数分析发现，OY-TES-1启动子甲基化水平与甲胎蛋白（AFP）含量呈负相。（2）通过构建不同截断型OY-TES-1启动子报告基因载体，转染细胞后，测双荧光素酶活性，确定了OY-TES-1核心启动子区域，进一步在体外将核心启动子告基因载体进行甲基化处理，发现OY-TES-1 核心启动子甲基化后其启动子活性明显下降，提示该区域可能存在甲基化调节的关键位点。（3）通过CHIP-qPCR证实转录因子Sp1可结合OY-TES-1核心启动子区，进一步干扰和过表达Sp1后，发现Sp1具有抑制OY-TES-1启动子活性的作用。（4）联合应用表观遗传学药物（DAC、VPA和TSA）能提高OY-TES-1在小鼠肝癌细胞的表达；将小鼠树突状细胞系DC2.4过表达OY-TES-1后可诱生小鼠CD8+T细胞为OY-TES-1特异的CTL；体外实验发现，OY-TES-1特异性CTL对表观遗传学药物干扰后的肝癌细胞杀伤能力明显提高；体内实验则显示，OY-TES-1 特异性CTL能缩小药物干扰后肝癌裸鼠移植瘤体积，抑制细胞增殖和促进细胞凋亡。这些结果提示： OY-TES-1的表达受甲基化调控，联合表观遗传学药物干预和OY-TES-1肝癌免疫治疗具有潜在的临床应用前景。