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。

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.