Hypoxia and long noncoding RNA

描述(申请人提供)：肿瘤细胞在肿瘤内低血管区域表达低氧诱导因子(HIF)以响应低氧。HIF激活了800多个基因，包括那些参与血管生成、糖酵解和生长因子信号转导的基因，这些基因共同促进了癌症的进展和转移。事实上，HIFs的表达增加是癌症患者预后不良的一个指标。此外，胚胎干细胞(ESCs)在低氧条件下比常氧条件下更有效地维持未分化状态。因此，了解HIFs的功能和调控对于肿瘤治疗和再生医学具有重要意义。目前的项目将在低氧培养的人乳腺癌细胞和胚胎干细胞(ESCs)中识别和表征与HIF-1结合的长非编码RNA(LncRNAs)。LncRNAs是指长度超过200个碱基且不编码mRNA、rRNA或tRNA的RNA。染色质免疫沉淀和RNA测序用于鉴定新的与HIF-1结合的LncRNAs(统称为R-HIF)，这些LncRNAs与糖酵解酶的编码基因有密切的联系。癌细胞和胚胎干细胞利用糖酵解作为葡萄糖的主要代谢途径，而不是在常氧条件下氧化磷酸化，这种趋势在低氧条件下被HIFs增强。增强糖酵解的优势是产生细胞快速增殖所需的更多代谢中间产物。因此，lncRNAs对糖酵解酶的调节可以作为癌症治疗的新靶点。目前，对lncRNAs在缺氧或糖酵解中的作用几乎一无所知。假设HIF-1利用lncRNAs作为新的共激活因子，在低氧条件下调节其靶基因，包括糖酵解基因，从而促进乳腺癌细胞和ESCs的增殖。为了检验这一假设，我们提出了以下三个目标。目的1旨在通过筛选R-HIF的DNA结合位点和鉴定其表达水平因R-HIF被敲除而改变的基因来鉴定R-HIF的直接靶基因。在目标2中，其他R-HIF在低氧基因调控中的作用将通过组合敲除、分析它们的全基因组结合部位和鉴定相关的染色质蛋白来确定。在目标3中，将通过能量和葡萄糖代谢的分析来确定R-HIFs在缺氧癌细胞和ESCs糖酵解增加和氧化磷酸化减少中的作用。这些研究有望建立lncRNAs作为一个新的实体，在低氧条件下HIF-1对糖酵解基因的调节是必不可少的。

DESCRIPTION (provided by applicant): Cancer cells express hypoxia-inducible factors (HIFs) in hypovascularized regions inside a tumor mass in response to hypoxia. HIFs activate more than 800 genes including those involved in angiogenesis, glycolysis, and growth factor signaling, which collectively facilitate cancer progression and metastasis. Indeed, increased expression of HIFs is an indicator for poor prognosis of cancer patients. In addition, embryonic stem cells (ESCs) maintain an undifferentiated state more efficiently in hypoxia than in normoxia. Therefore, it is important to understand the functions and regulation of HIFs for cancer therapy and regenerative medicine. The current project will identify and characterize long noncoding RNAs (lncRNAs) that are bound to HIF-1 in human breast cancer cells and embryonic stem cells (ESCs) cultured in hypoxia. LncRNAs are defined as RNAs that are longer than 200 bases and do not encode mRNA, rRNA, or tRNA. Chromatin immunoprecipitation and RNA sequencing were used to identify novel HIF-1-bound lncRNAs (collectively called R-HIFs) that have been mapped closely to genes encoding glycolysis enzymes. Cancer cells and ESCs use glycolysis as a primary metabolic pathway of glucose rather than oxidative phosphorylation in normoxia and this tendency is enhanced by HIFs in hypoxia. Enhanced glycolysis has an advantage of producing more metabolic intermediates necessary for rapid cell proliferation. Thus, the regulation of glycolytic enzymes by lncRNAs can serve as a novel target for cancer therapy. Currently virtually nothing is known about the involvement of lncRNAs in hypoxia or glycolysis. It was hypothesized that HIF-1 uses lncRNAs as novel coactivators to regulate its target genes, including glycolysis genes, in hypoxia and thereby promotes proliferation of breast cancer cells and ESCs. The following 3 aims have been proposed to test this hypothesis. Aim 1 is designed to identify direct target genes of an R-HIF by screening of its DNA binding sites and by identification of genes whose expression level is altered by the knockdown of the R-HIF. In Aim 2 the roles of other R-HIFs in hypoxic gene regulation will be determined by the combination of knockdown, analysis of their genome-wide binding sites, and identification of associated chromatin proteins. In Aim 3 the roles of the R-HIFs in increased glycolysis and decreased oxidative phosphorylation in hypoxic cancer cells and ESCs will be determined through the analysis of energy and glucose metabolism. These studies are expected to establish lncRNAs as a novel entity essential for the regulation of glycolytic genes by HIF-1 in hypoxia.