罕见肿瘤家系中克隆的候选瘤基因TILZ4a致瘤机制研究

Supported by our recently completed grant from the National Natural Science Foundation of China, we investigated a rare, large multi-tumor pedigree by genome-wide linkage analysis using SNP chips and whole exome next-generation sequencing. We identified a causative T/C mutation in the 5'-untranslated region (5'-UTR), 90 bp before the start codon of the TILZ4a gene, which is located on chromosome 3q25.1. The C mutation allele co-segregated with the disease phenotype, and our functional study implied that this mutant allele may cause TILZ4a gene up-regulation in members of this multi-tumor pedigree through binding site depletion of a transcriptional suppressor and transcriptional inhibition escape. Our group and others demonstrated that the TILZ4a gene was up-regulated in several types of tumor biopsies compared to their normal tissue counterparts. These results indicated that TILZ4a may act as an oncogene. Bioinformatic prediction also implied that TILZ4a is likely a transcription factor. However, there are no publications reporting a tumorigenesis mechanism for this gene. In this proposal, we intend to screen and validate proteins interacting with TILZ4a using yeast two-hybrid and other assays, identify its downstream regulated genes by capturing its binding sites using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). After we determine the TILZ4a interacting and downstream proteins, we will systematically and comprehensively elucidate the main signaling pathways involving TILZ4a that play a vital role in its tumorigenesis in vitro. Finally, we will also construct a transgenic mouse model that exogenously expresses the TILZ4a gene to validate its tumorigenesis effect and mechanisms in vivo. Completing the goals of this proposal will strengthen our understanding of this novel oncogene and provide a new target for cancer prevention and personal treatment.

在上一国家自然科学基金资助下，我们对一个罕见多肿瘤大家系进行了全基因组连锁分析，结合新一代测序技术进行全外显子组测序，鉴定出了染色体3q25.1区域TILZ4a基因翻译起始位点上游-90位点的T/C突变与疾病表型共分离，功能研究表明该突变改变了一个转录抑制因子结合位点而使家系携带者TILZ4a基因表达上调，我们在多种肿瘤标本中也证实了该基因表达上调，提示TILZ4a可能是一个瘤基因，生物信息学预测它可能是一个核转录因子，但目前尚无人对它的致瘤功能及机制进行研究。本项目拟在体外通过酵母双杂交等技术寻找其相互作用蛋白，用染色质免疫沉淀结合新一代测序技术捕获其转录调控的下游基因，并深入探讨TILZ4a在癌变过程中参与的主要信号通路，最后在转基因小鼠体内进一步验证其成瘤作用及机制。本项目的完成可以诠释TILZ4a这一全新候选瘤基因的致瘤机制，同时可为肿瘤的早期预防和个体化治疗等提供新的分子靶标。

我们前期采集到一个罕见的多肿瘤高发大家系，该家系6代103个成员中有15人先后罹患肿瘤，发病谱包括结直肠癌、乳腺癌、乳腺纤维瘤、子宫内膜癌、子宫肌瘤、胃癌、血管瘤等，其中一些患者甚至先后在不同部位发生多种肿瘤。遗传连锁分析将致病基因定位于染色体3q24-26区域，新一代测序技术对家系中3个代表性成员进行全外显子组测序筛查，全部24个家系核心成员中验证发现定位于染色体3q25.1区域的TSC22D2(原名TILZ4a，现GenBank中标准名称为TSC22D2，故本结题报告统一用最新标准名称)是该家族发病的致病基因。本项目对该基因在肿瘤发生发展中的作用机制进行了研究。.TSC22D2编码的蛋白是TSC-22/Dip/Bun家族的一员。有关TSC22D2基因功能的研究还很少，在恶性肿瘤发生发展中的作用可以说还完全不清楚。我们通过在线芯片的数据分析TSC22D2在不同类型恶性肿瘤中的表达情况，初步检测TSC22D2对肺癌细胞系A549生长的调节作用。酵母双杂交技术寻找与TSC22D2相互作用的蛋白，筛选到的44个TSC22D2交互作用蛋白中挑选了RNF2和WDR77进行免疫共沉淀和荧光共定位等实验验证。证实了TSC22D2与WDR77之间存在交互作用，发现WDR77能够抑制TSC22D2对于A549细胞生长的负性调节作用，为TSC22D2在肿瘤发生发展中的功能研究提供了线索。.GEO中的表达谱芯片数据发现TSC22D2在结直肠癌组织中表达也明显下调，随后我们在结直肠癌临床样本中进行验证； TSC22D2能明显抑制肿瘤细胞的生长，提示TSC22D2具有抑瘤基因特性。免疫共沉淀结合质谱分析技术共筛选到142个TSC22D2相互作用蛋白，并从中挑选了一个与肿瘤细胞生长相关的蛋白PKM2进行验证，过表达TSC22D2可影响核内PKM2的水平，并下调PKM2下游基因cyclin D1的表达，这可能是TSC22D2抑制肿瘤细胞生长的机制之一。