组蛋白修饰调节TCF4 选择性剪接在食管癌发生发展中的作用机制研究

Wnt/β-catenin signaling pathway consists of some transcription coactivators such as β-catenin and T cell factor (TCF). This signaling pathway has been recognized to control a variety of functions and properties in various types of cells. During tissue development and regeneration, Wnt signals ensure the proper balance between proliferation and differentiation. Activation of Wnt/β-catenin signaling pathway is thought to be associated with cell proliferation, as aberrant expressions of Wnt components are found in many types of cancers. In our preliminary study, we found there were various types of splicing forms of TCF4 in esophageal carcinoma cell line EC109, which indicated TCF4 mRNAs were alternative spliced during its maturation. Result of western blotting showed two main proteins isoforms of 55 and 75kd were found in this cell line. Interestingly, its protein isoform of 55kd was efficiently down-regulated by TSA treatment or by HDAC1 knockdown via siRNA, which has no effect on the other isoform. Moreover, RT-PCR analysis showed the mRNA isoforms were also deregulated by TSA treatment. Our preliminary data suggested alternative splicing of TCF4 mRNA was regulated by histone modification, as TSA treatment and HDAC1 knockdown led to hyperacetylation of core histone. In this study, we determine to fully discover all the mRNA splicing forms in ESCC cancer and cell lines, and to identify the cis elements in the genomic of TCF4 which affect alternative splicing, and to elucidate the mechanism how to regulate TCF4 splicing by histone modification and finally to demonstrate the function of individual isoform. This study helps to comprehensively understand the biological functions of tumor relative gene TCF4 in ESCC cancer.

选择性剪接是后基因组时代的重要研究内容。TCF4是Wnt/Catenin信号途径的组成部分，参与肿瘤发生发展。研究表明：TCF4 mRNA存在选择性剪接，在蛋白水平主要表现为具有CtBP结构域的75Kd(抑癌)和缺失该区域的55Kd(促癌)两种形式。在前期工作中，我们发现干扰HDAC1基因表达或抑制HDAC活性后，食管癌细胞中TCF4短链蛋白明显地被抑制，而长链蛋白无变化，且mRNA表达形式也发生改变。由于HDAC影响染色质组蛋白修饰，因此推测TCF4基因的组蛋白修饰可能影响着其转录后选择性剪接，从而影响着食管癌的发生发展。因此本项目拟以食管癌细胞株及临床样品为对象，确定TCF4基因的剪接形式与组蛋白修饰的对应关系，分析组蛋白修饰调控TCF4 mRNA剪接的分子机制，确定参与TCF4剪接的反式作用因子，并确定其选择性剪接体的功能，从而揭示TCF4基因选择性在食管癌发生发展中的生物学意义。

择性剪接是后基因组时代的重要研究内容。TCF4是Wnt/Catenin信号途径的组成部分，参与肿瘤发生发展。我们前期的研究表明：TCF4 mRNA存在选择性剪接，在蛋白水平主要表现为具有CtBP结构域的75Kd(抑癌)和缺失该区域的55Kd(促癌)两种形式。 前期工作还表明：干扰HDAC1基因表达或抑制HDAC活性后，食管癌细胞中TCF4短链蛋白明显地被抑制，而长链蛋白无变化，且mRNA表达形式也发生改变。由于HDAC影响染色质组蛋白修饰，我们分析认为TCF4基因的组蛋白修饰可能影响着其转录后选择性剪接，从而影响着食管癌的发生发展。因此在本项目中，我们以食管癌细胞株及临床食管癌肿瘤组织样品为对象，确定了TCF4基因的各种mRNA剪接形式，新发现只表达N端结构域的截短型剪接体异构体，在蛋白水平上分析了M和E型TCF4的表达水平及其与组蛋白修饰的关系，并阐明了两种不同大小分子的蛋白异构体在食管癌细胞中的生物学功能。为探索食管癌细胞在转录组水平的mRNA剪接形式的改变，采用RNA-Seq技术分析TSA处理前后细胞的差异剪接形式改变，提供了进一步研究的靶点。我们还采用CoIP方法分析了TCF4的相互作用蛋白，并对TCF4与HDAC1的相互作用进行了探索。在此基础上初步分析TCF4 mRNA剪接的分子机制，但未获得理想的结果。本研究表明TCF4基因选择性剪接在食管癌中存在复杂的过程，各种不同异构体的生物学功能各不相同。本研究对于揭示食管癌的发生发展的分子机制具有一定的意义。