Role of hypoxia in altering hnRNP A2B1-mediated interaction with m6A to promote breast cancer progression

项目摘要/摘要 背景：N6-甲基腺苷(M6A)是真核生物中最普遍的转录后修饰 通过调节选择性剪接和转录调控，在基因表达调控中发挥重要作用 RNA周转率。最近的研究表明，M6A残基的失调与乳腺癌的发生和发展有关 进展，特别注意到低氧应激导致相关的m6A水平显著降低 转移性表型总体增加。低氧也与异常的选择性剪接有关 然而，在乳腺癌中，这种关系是如何调节的仍有待阐明。值得注意的是，RNA结合 MRNA前加工蛋白hnRNP A2/B1的能力受到m6A残基的影响，该蛋白具有 也与乳腺癌细胞的侵袭和迁移有关。A2/B1和M6A都参与了 对选择性剪接的调节，我们的初步数据表明，m6A修饰直接抑制 A2/B1结合。因此，低氧时m6A水平降低可能导致A2/B1活性显著升高。 目标/假设：该建议假设由于低氧应激导致m6A残基的耗尽 导致肿瘤相关基因hnRNP A2/B1结合增加，导致RNA剪接异常 以及随之而来的乳腺癌进展。这一想法将通过描述两国关系的特征来研究。 低氧乳腺癌细胞A2/B1活性与M6A水平的关系及M6A差异的判定 沉积可以影响A2/B1介导的选择性剪接，促进乳腺癌的表型。 具体目的：研究低氧对乳腺癌M6A：A2/B1介导的RNA剪接的影响。 缺氧诱导的M6A：A2/B1依赖的亚型将在常氧癌细胞中鉴定和表达，以 评估它们是否可以调节癌症表型。为了刻画M6A的分子机制 在缺氧过程中残基改变A2/B1结合，一种改进的CLIP策略将被用于识别m6A-depeden A2/B1结合位点随后将被修改以确认残基是直接调节A2/B1结合的。 培训计划/环境：为了实现我成为一名独立癌症研究员的目标，我有 制定了由三个具体目标组成的培训计划：1)学习新的分子/癌症生物学和 计算方法，2)提高我的科学写作和口语交流能力，3)发展 一个由同事和导师组成的专业网络。在密歇根大学培育的培训环境 科罗拉多州AMC拥有独特的设备，可以通过维护最先进的设施来帮助我实现这些目标 并资助与我对RNA和癌症研究的兴趣直接一致的倡议。 与健康相关：NCI最近将RNA生物学描述为分子领域中最有影响力的领域之一 医药。通过研究低氧对m6A介导的乳腺hnRNP A2/B1 RNA剪接活性的影响 癌症，这一提议将为这种致命疾病的“表位编码组”提供新的见解，并可能确定 RNA介导的癌症进展途径的新治疗靶点。

Project Summary / Abstract Background: N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and plays an important role in mediating gene expression through regulation of alternative splicing and RNA turnover. Recent studies have implicated dysregulation of m6A residues in breast cancer initiation and progression, specifically noting that hypoxic stress causes a significant reduction of m6A levels that is correlated with an overall increase in metastatic phenotypes. Hypoxia has also been linked to aberrant alternative splicing in breast cancer, though how this relationship is mediated remains to be elucidated. Notably, the RNA binding ability of the pre-mRNA processing protein hnRNP A2/B1 is influenced by m6A residues, and this protein has also been implicated in the invasion and migration of breast cancer cells. Both A2/B1 and m6A are involved in the regulation of alternative splicing, and our preliminary data suggests that m6A modifications directly inhibit A2/B1 binding. Therefore, reduced m6A levels during hypoxia may cause a significant increase in A2/B1 activity. Objective / Hypothesis: This proposal hypothesizes that depletion of m6A residues due to hypoxic stress causes an increase in hnRNP A2/B1 binding of cancer-associated genes leading to dysregulated RNA splicing and subsequent breast cancer progression. This idea will be investigated by characterizing the relationship between A2/B1 activity and m6A levels in hypoxic breast cancer cells and determining whether differential m6A deposition can influence A2/B1-mediated alternative splicing and promote breast cancer phenotypes. Specific Aims: To examine how hypoxia influences m6A:A2/B1-mediated RNA splicing in breast cancer, hypoxia-induced m6A:A2/B1-dependent isoforms will be identified and expressed in normoxic cancer cells to evaluate whether they can mediate a cancer phenotype. To characterize the molecular mechanism of how m6A residues alter A2/B1 binding during hypoxia, a modified CLIP strategy will be used to identify m6A-depedent A2/B1 binding sites that will then be modified to confirm the residues are directly modulating A2/B1 binding. Training Plan / Environment: To achieve my goal of becoming an independent cancer investigator, I have developed a training plan composed of three specific objectives: 1) to learn new molecular/cancer biology and computational approaches, 2) to improve my scientific writing and oral communication skills, and 3) to develop a professional network of colleagues and mentors. The training environment fostered at the University of Colorado AMC is uniquely equipped to help me achieve these goals through maintaining state-of-the-art facilities and funding initiatives that are directly aligned with my interest in RNA and cancer research. Health Relevance: The NCI recently characterized RNA biology as one of the most influential areas in molecular medicine. By investigating how hypoxia influences m6A-mediated hnRNP A2/B1 RNA splicing activity in breast cancer, this proposal will provide novel insights into the ‘epitranscriptome’ of this deadly disease and may identify novel therapeutic targets of RNA-mediated cancer progression pathways.