SVIL-HIF1α信号轴调控缺氧微环境诱导的肝癌转移机制研究

The tumor microenvironment is widely recognized as a key player in the promotion of tumor growth and malignant progression. Hypoxia is a major phenomenon in all solid tumour microenvironments. HIF1α (Hypoxia-inducible factor 1 alpha) is a primary regulator of the transcriptional response to hypoxia by regulating large numbers of genes involved in glycolysis, cell survival, proliferation, invasion and metastasis. The function of HIF1α was affected by protein stability, mRNA translation and gene transcription in hypoxia. We found three different splicing isoforms of SVIL (Supervillin), SV1/SV4/SV5, exist in tumour cells. Among of them, SV4 and SV5 protein levels linearly increase in accord with HIF1α to hypoxia. Our recently data shows SVIL is an upstream regulator of HIF1α through enhancing its transcription, thereby to regulate epithelia to mesenchymal transition (EMT) and angiogenesis in hypoxia. However, it is still very limited to the understanding of molecular mechanism of SVIL to regulate the transcription of HIF1α and enhance tumour metastasis by SVIL-HIF1α axis in hypoxia microenvironment. We propose to combine molecular biology, cell biology and experimental animal model to further dissect the properties of SVIL-HIF1α axis in regulation of HCC metastasis in hypoxia. Our aim is to identify SVIL interaction partners in hypoxia and reveal the regulatory effect by the interaction network, the signaling pathway and epigenetic modification in regulation of HIF1α transcription. Furthermore, we try to explore the pathological mechanism of the disease resulted from the abnormal expression of SVIL in hypoxia, which will provide theoretical basis for intervention of diseases and lay out a solid foundation for future clinical research.

肿瘤微环境是促进肿瘤生长与转移过程的关键调控因素，缺氧微环境是实体瘤常见现象。HIF1α是主要缺氧调节因子，通过增强相关基因转录，影响肿瘤代谢、存活、增殖、侵袭与转移，HIF1α功能受到蛋白稳定性、mRNA翻译、与转录水平调控。研究发现SVIL存在三个剪切异构体，SV1/SV4/SV5。缺氧上调SV4/SV5表达，与HIF1α呈相同趋势。我们最新结果显示SVIL是HIF1α上游调节因子，通过HIF1α促进上皮-间质细胞转换、新生血管生成等。但缺氧微环境引起SVIL促HIF1α转录、SVIL-HIF1α信号轴促肿瘤转移的作用机制仍不明了。我们拟采用分子、细胞与实验动物等手段，进一步解析缺氧诱导SVIL-HIF1α信号轴促肝癌转移的机制；揭示缺氧环境下SVIL相互作用网络、信号传导途径、表观修饰增强HIF1α转录方式；探索缺氧下SVIL表达异常的病理机制，为相关疾病干预与临床研究提供理论基础。

肿瘤微环境是促进肿瘤生长与转移过程的关键调控因素，缺氧微环境是实体瘤常见现象。HIF1α是主要缺氧调节因子，通过增强相关基因转录，影响肿瘤代谢、存活、增殖、侵袭与转移，HIF1α功能受到蛋白稳定性、mRNA翻译、与转录水平调控。但缺氧微环境中如何通过SVIL-HIF1α信号轴促肿瘤发生与转移的机制不明了。我们综合应用细胞生物学、生物化学、分子生物学、动物模型等技术，利用质谱鉴定SVIL体内互作网络与蛋白质修饰。首先发现了一个新的SVIL剪切异构体SV5，在组织与细胞水平都表达，与其它异构体之间存在功能冗余性，促进细胞的增殖与迁移。进而发现SVIL在肝癌组织中表达显著上调，且与肿瘤缺氧微环境相关，我们结果显示SVIL是HIF1α上游调节因子，通过HIF1α促进上皮-间质细胞转换、新生血管生成等。具体作用表现为SVIL与小分子GTPase包括RhoA、Cdc42、Rac1互作，促进其RhoA激活，并通过RhoA-ROCK信号通路传递ERK/p38信号，导致EMT相关基因表达变化，增强肝癌细胞的在体内外的转移潜能。同时，SVIL也促进肝细胞癌的血管生成，SVIL在内皮细胞血管与血管拟态结构上都有定位，影响内皮细胞和肝癌细胞的体外成管能力，可能是通过促血管生长因子VEGF的胞外分泌影响内皮细胞血管和血管拟态的形成。SVIL促进肝癌进展与血管生成、信号传递，细胞增殖密切联系，我们新发现其直接参加细胞周期的调控而促进肿瘤细胞的增殖能力，主要在细胞有丝分裂期SVIL受到CDK1磷酸化，招募中心体关键蛋白AuroraA，保证有丝分裂纺锤体组装与染色体分离，从而促进细胞增殖进程。此外，以斑马鱼与小鼠为模型，通过基因敲除的方法，发现抑制svila 基因的表达影响斑马鱼胚胎的集中延伸运动。完全基因敲除成年小鼠没有表达明显异常，但可能与发育迟缓现象有关，为下一步研究打下了基础。这些研究结果揭示缺氧环境下SVIL相互作用网络、信号传导途径、翻译后修饰，增强HIF1α表达提供了理论基础；也为SVIL表达异常在肝癌发生发展进程中的病理机制，与疾病干预提供理论支撑。