核呼吸因子1调控HIF-1α表达对细胞低氧适应的影响

Hypoxia is a common pathological process for mamy diseases, and one of the most frequent medical problem in specific environment. The nuclear respiratory factor-1 (NRF-1)serves as a transcription factor that activates the expression of a wide range of nuclear genes essential for mitochondrial biogenesis and function, including mitochondrial respiratory complex subunits.Recent research identified the existence of the NRF-1 binding consensus sequence in the proximal promoter regions of more than two thousand genes, suggesting more potential function of NRF-1 might be discovered. Hypoxia-inducible factor 1 (HIF-1), a most important transcription factor regulating more than one hundred target genes under hypoxic conditon, has long been a research focus to study hypoxic adaptation.However,the regulation of HIF-1α in transcriptional level has not been fully elucidated. We first discovered the functional binding sites in the 5'-regulator region of HIF-1α and the negtive regulation of HIF-1α by NRF-1 in HEK293T cell line. We also observed the different expression of NRF-1 between normal tissue and carcinoma cells under hypoxia, which negtively correlated to HIF-1α mRNA levels. The project will focus on the regulation of HIF-1α expression by NRF-1 and the effect on cellular adaptation to hypoxia. This work will suggest a novel mechanism for hypoxic adaptation,supplying a new suitable solution to study the various response to hypoxia between normal and cancer cells.

低氧是许多疾病共有的病理过程，也是特殊环境最常见的医学问题之一。核呼吸因子1（NRF-1）是调控线粒体呼吸链蛋白表达的核转录因子，最新研究显示在二千余种基因的启动子区含有NRF-1结合共有序列，提示NRF-1有更多的潜在功能。低氧诱导因子1（HIF-1）是研究低氧适应最受关注的热点，但是对HIF-1α转录水平调控的了解还非常有限。我们前期研究在HEK293T细胞中首次发现NRF-1功能性结合于人HIF-1α基因5'-调控区，对HIF-1α基因表达具有负调控作用。低氧对正常组织细胞和肿瘤细胞的NRF-1具有不同影响，且NRF-1蛋白水平与HIF-1α mRNA 水平负相关。故本项目拟系统研究NRF-1对HIF-1α基因表达的调控作用，探讨该调控作用对细胞低氧适应的影响。该研究将提出细胞低氧适应的新机制，并为研究正常与肿瘤细胞在低氧适应中的不同反应提供新的切入点。

低氧是许多疾病共有的病理过程，也是特殊环境最常见的医学问题之一。核呼吸因子1（NRF-1）是调控线粒体呼吸链蛋白表达的核转录因子，最新研究显示在二千余种基因的启动子区含有NRF-1结合共有序列，提示NRF-1有更多的潜在作用。低氧诱导因子1（HIF-1）作为调控低氧下百余种基因表达的转录因子，是研究低氧适应最受关注的热点。.本项目发现HIF-1α基因5’-调控区存在两个具有功能的NRF-1结合位点，其核心序列分别位于-14 ~ -10和+23 ~ +27。NRF-1对HIF-1α基因有负调控作用，具有种属保守性，且不依赖于TATA 盒结合蛋白的参与。这种负调控机制在低氧下仍旧运作。我们还观察到低氧下不同器官组织、正常细胞和肿瘤细胞中NRF-1的表达变化存在不一致性。同时比较正常细胞和肿瘤细胞低氧适应性方面，我们发现正常细胞与肿瘤细胞也不一致。低氧下正常细胞中NRF-1通过对HIF-1α的调控作用，继而影响HIF-1α下游的与低氧适应相关的靶基因，如LDHA，HK-1和GLUT1。但在肿瘤细胞中，NRF-1虽然仍能负调控HIF-1α，却不能影响这些低氧适应相关的下游靶基因表达。除此之外，我们收集大量临床肝癌患者的病理组织样本，进行免疫组化和统计分析，非肿瘤和肿瘤患者肝脏组织切片中细胞核内NRF-1表达水平低于非肿瘤组，核内HIF-1α表达水平高于非肿瘤组，具有统计学差异，而胞质内HIF-1α的表达水平不具有差异。细胞核内NRF-1和细胞质内HIF-1α的相关系数为负，表明两者呈负相关。核HIF-1α表达高低与肿瘤TNM分期、直径密切相关，同时核NRF-1表达高低与血管侵袭、肿瘤TNM分期和肝硬化密切相关。细胞核内NRF-1高表达的肝细胞癌患者，其五年生存率高于低表达组；细胞核内HIF-1α高表达的肝细胞癌患者，其五年生存率高于核内HIF-1α低表达组，而胞质内HIF-1α高表达的肝细胞癌患者，其五年生存率低于胞质HIF-1α低表达组。.本研究提出了NRF-1通过调控HIF-1α影响细胞低氧适应的新机制。同时也发现正常与肿瘤细胞中NRF-1在低氧适应中的具有不同反应，提示我们正常细胞和肿瘤细胞中NRF-1和HIF-1α的调控机制可能存在不同，可为将来研究低氧适应提供新的切入点。