Hypoxia and long noncoding RNA

描述（由申请人提供）：癌细胞在肿瘤块内的低血管化区域表达缺氧诱导因子（HIF），以响应缺氧。HIF激活800多个基因，包括参与血管生成、糖酵解和生长因子信号传导的基因，这些基因共同促进癌症进展和转移。事实上，HIF的表达增加是癌症患者预后不良的指标。此外，胚胎干细胞（ESCs）在缺氧中比在常氧中更有效地维持未分化状态。因此，了解HIF的功能和调控对肿瘤治疗和再生医学具有重要意义。目前的项目将鉴定和表征在缺氧条件下培养的人乳腺癌细胞和胚胎干细胞中与HIF-1结合的长链非编码RNA（lncRNA）。LncRNA被定义为长度超过200个碱基并且不编码mRNA、rRNA或tRNA的RNA。染色质免疫沉淀和RNA测序用于鉴定新的HIF-1结合的lncRNA（统称为R-HIF），这些lncRNA与编码糖酵解酶的基因密切相关。癌细胞和ESC使用糖酵解作为葡萄糖的主要代谢途径，而不是在常氧下的氧化磷酸化，并且这种趋势在缺氧下被HIF增强。增强的糖酵解具有产生快速细胞增殖所必需的更多代谢中间体的优点。因此，通过lncRNA调节糖酵解酶可以作为癌症治疗的新靶点。目前，几乎没有什么是已知的lncRNA参与缺氧或糖酵解。据推测，HIF-1使用lncRNA作为新的共激活因子来调节其靶基因，包括糖酵解基因，从而促进乳腺癌细胞和ESC的增殖。以下三个目标被提出来检验这一假设。目的1旨在通过筛选R-HIF的DNA结合位点和通过鉴定其表达水平因R-HIF的敲低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敲低、分析其全基因组结合位点和鉴定相关染色质蛋白的组合来确定。在目标3中，R-HIF在缺氧癌细胞和ESC中增加糖酵解和减少氧化磷酸化中的作用将通过能量和葡萄糖代谢的分析来确定。这些研究有望建立lncRNA作为一种新的实体的糖酵解基因的调控HIF-1在缺氧。

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.