药靶候选基因CHMP4A参与缺氧诱导因子1调控的功能及机制研究

HIF-1 (hypoxia-inducible factor-1) is the transcription factor that is most closely associated with hypoxia. HIF-1 is overexpressed in most solid tumors and plays a vital role in hypoxic acclimatization, energy metabolism, tumor angiogenesis, tumor invasion, and drug tolerance in cancer cells. To identify novel human genes associated with the stability and transcriptional activity of HIF-1, a cell-based dual luciferase reporter system based on a HRE-LUC (hypoxia responsive element luciferase) reporter gene was constructed to screen 409 novel human genes cloned in our lab. We found that CHMP4A (chromatin modifying protein 4A) can significantly up-regulate the HRE-LUC activity under both normoxic and hypoxic environment. CHMP4A belongs to the chromatin-modifying protein/charged multivesicular body protein (CHMP) family. These proteins are components of ESCRT-Ш (endosomal sorting complex required for transport Ш). Our results demonstrated that CHMP4A can increase /stable the expression of HIF-1α protein, and enhance the transcription level of VEGF and AK3, downstream target genes of HIF-1. This project will further study the biology function of CHMP4A, and the regulating pathways or mechanisms on HIF-1, as well as the effect on cardiovascular diseases and tumor hypoxia regulation. These will be the foundation for the clinical application of CHMP4A as a potential drug target.

缺氧诱导因子1（HIF-1）是迄今发现与缺氧关系最为密切的转录因子，HIF-1在大多数实体瘤中过表达，在缺氧适应，能量代谢，肿瘤血管生成，肿瘤侵袭，及癌细胞耐药性等方面起着至关重要的作用。为了寻找HIF-1活性调节基因，构建了基于缺氧反应原件的荧光素酶报告系统（HRE-Luc），通过对自主克隆的409个人类基因进行筛选，发现CHMP4A基因在常氧及缺氧条件下都能增强HRE-luc报告基因活性。人CHMP4A属于染色质修饰蛋白/带电荷多泡体蛋白家族成员，是转运必需内吞体分选复合物Ш的主要组份。预实验表明CHMP4A能增加/稳定HIF-1α的蛋白表达，并增强HIF-1下游靶基因VEGF和AK3的转录活性。本项目将进一步研究CHMP4A的生物学功能，探讨其参与HIF-1调控的途径和分子机制，以及在心脑血管疾病和肿瘤缺氧调节中的作用，为CHMP4A作为潜在药物靶标的临床应用奠定基础。

缺氧诱导因子1（HIF-1）是迄今发现与缺氧关系最为密切的转录因子，HIF-1在大多数实体瘤中过表达，在缺氧适应，能量代谢，肿瘤血管生成，肿瘤侵袭，及癌细胞耐药性等方面起着至关重要的作用。为了寻找HIF-1活性调节基因，构建了基于缺氧反应原件（HRE）的HRE-Luc荧光素酶报告系统，通过对自主克隆的409个人类基因进行筛选，发现CHMP4A基因在常氧及缺氧条件下都能增强HRE-luc报告基因活性。人CHMP4A属于染色质修饰蛋白/带电荷多泡体蛋白家族成员，是转运必需内吞体分选复合物III的主要组份。实验表明CHMP4A在肺癌细胞系中较高表达，在前列腺癌和乳腺癌中CHMP4A的表达与HIF1A明显负相关，CHMP4A 在相关肿瘤组织中高表达很有可能是个好的诊断标志物。功能实验表明CHMP4A能增加/稳定HIF-1α的蛋白表达，并增强HIF-1下游靶基因VEGF和AK3的转录活性。进一步过表达及敲除实验研究发现CHMP4A对于HIF-1α活性及其相关靶基因VEGF、MMP2和c-MET的表达都有调节作用，CHMP4A可促进细胞增殖及克隆形成能力。体内实验的初步结果也显示CHMP4A对于肺癌的发生发展有促进作用，为CHMP4A作为潜在药物靶标的临床应用奠定基础。