Examining the Role of a Novel Long Noncoding RNA, linc02454, in Resistance of Glioblastoma to Temozolomide

项目摘要/摘要 摘要脑胶质母细胞瘤(Gbm)是最常见的脑部原发肿瘤，由于不可避免，患者的生存期为15个月。 尽管采用标准治疗-替莫唑胺化疗(TMZ)、放射治疗和手术，肿瘤仍可复发。 确定和靶向调控GBM化疗耐药的基因非常关键。长的非编码RNA LncRNAs是一类新的基因转录本，占基因组的80%。这些lncRNA诱导 转录调控和表型的深刻变化，包括化疗反应。因为 很少有人研究过GBM lncRNA，在这一丰富而新颖的类别中描述了新的候选基因 基因可能会显著提高化疗效果。评估参与GBM的lncRNAs 化疗耐药，我们确定linc02454是TMZ后表达最高的lncRNAs 治疗。TMZ诱导的linc02454基因敲除(Kd)降低了GBM细胞的存活率，而RNA- SEQ发现CXC-趋化因子受体4(CXCR4)是继 IncRNA KD。CXCR4是一种特性良好的GBM化疗耐药调节剂，目前靶向为多个 临床试验。我们假设TMZ调节linc02454的表达，并且linc02454对GBM产生抗性 通过CXCR4的调制到TMZ。在本项目中，我们将评估linc02454在GBM响应中的作用 接受TMZ治疗。在目标1中，我们确定linc02454是否依赖于CXCR4表达的增加，评估 上位性和CXCR4救援的功能相互作用，以及评估linc02454是否直接 通过荧光原位杂交调控CXCR4。在目标2中，我们将评估TMZ如何增加 Linc02454。SA2.1将对DNA基因座(包括linc02454)上的lncRNA甲基化进行综合分析 初次切除/复发时依赖甲基化的lncRNA转录。SA2.2将评估染色质状态 TMZ前后的linc02454。在目标3中，我们将评估当与TMZ结合时，linc02454是否抑制 +放射治疗，提高体外和体内的治疗反应。这些目标将检验lin02454是否 思考GBM通过CXCR4对TMZ的反应以及TMZ暴露如何调节linc02454。我们还将 检测linc02454是否是GBM治疗的潜在靶点。如果成功，这项为期五年的研究将 在TMZ诱导的治疗耐药性的背景下，提供对lncRNA生物学的更彻底的理解。 在我的导师小组的帮助下，我们制定了一项职业发展计划，用于在 GBM lncRNAs转录调控。该项目将通过定期一对一的方式进行密切监督 与初级导师Dou博士的互动，他是染色质和转录调控方面的国际专家 在癌症中。NIH资助的联合导师，专注于GBM生物学的神经外科医生Behnam Badie博士和Dr。 威廉·麦晋桁也有丰富的经验，指导神经外科医生和科学家走向独立，并 发挥亲密导师的作用。重要的是，通过完成这项工作，我将发展转录方面的专业知识 调节，lncRNAs功能的一个关键组成部分，以及治疗引起的GBM的变化。

Project Summary/Abstract Glioblastoma (GBM), the most common primary brain tumor, has a 15 month patient survival due to inevitable recurrence of tumor despite standard treatment – temozolomide chemotherapy (TMZ), radiation, and surgery. It is critical to characterize and target genes modulating GBM chemoresistance. Long noncoding RNAs (lncRNAs) are a novel class of genetic transcripts comprising 80% of the genome. These lncRNAs induce profound alterations in transcriptional regulation and phenotype, including chemotherapy response. Because few GBM lncRNAs have been studied, characterizing new candidates among this abundant and novel class of genes may significantly improve chemotherapeutic efficacy. To assess lncRNAs involved in GBM chemotherapy resistance, we identified linc02454 among the most highly upregulated lncRNAs following TMZ treatment. Knockdown (KD) of linc02454 decreased in vitro GBM cell viability in response to TMZ, while RNA- seq identified CXC-chemokine receptor type 4 (CXCR4) among the most heavily downregulated genes after lncRNA KD. CXCR4 is a well-characterized modulator of GBM chemoresistance, currently targeted in multiple clinical trials. We hypothesize TMZ regulates linc02454 expression and that linc02454 exerts GBM resistance to TMZ through modulation of CXCR4. In this project, we will evaluate the role of linc02454 on GBM response to TMZ treatment. In aim 1, we identify if linc02454 relies upon increased CXCR4 expression, assessing functional interaction via epistasis and CXCR4 rescue, as well as evaluating whether linc02454 directly regulates CXCR4 via Fluorescence in Situ Hybridization. In aim 2, we will assess how TMZ increases linc02454. SA2.1 will perform integrative analysis of lncRNA methylation at DNA loci (including linc02454) and methylation-dependent lncRNA transcription at initial resection/recurrence. SA2.2 will assess chromatin state of linc02454 before and after TMZ. In aim 3, we will assess if linc02454 repression, when combined with TMZ + radiotherapy, improves treatment response, in vitro and in vivo. These aims will examine whether lin02454 meditates GBM response to TMZ via CXCR4 and how linc02454 is regulated by TMZ exposure. We will also test whether linc02454 is a potential therapeutic target in GBM therapy. If successful, this five-year study will provide a more thorough understanding of lncRNA biology in the context of TMZ-induced treatment resistance. With my mentorship group, we have developed a career development plan for tailored training in transcriptional regulation of GBM lncRNAs. The project will be closely supervised through regular one-on-one interaction with primary mentor Dr. Yali Dou, an international expert in chromatin and transcriptional regulation in cancer. NIH-funded co-mentors, Dr. Behnam Badie, a neurosurgeon focusing on GBM biology, and Dr. William Mack also have extensive experience mentoring neurosurgeon-scientists to independence, and have functioned as close mentors. Importantly, by completing this work, I will develop expertise in transcriptional regulation, a critical component in the function of lncRNAs and treatment-induced changes in GBM.