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

项目概要/摘要 胶质母细胞瘤（GBM）是最常见的原发性脑肿瘤，由于不可避免的肿瘤转移， 标准治疗-替莫唑胺化疗（TMZ）、放疗和手术后肿瘤复发。 关键是表征和靶向调节GBM化学抗性的基因。长非编码rna lncRNA是一类新的基因转录物，占基因组的80%。这些lncRNA诱导 转录调控和表型的深刻改变，包括化疗反应。因为 已经研究了一些GBM lncRNA，在这一丰富而新颖的类别中表征了新的候选者。 基因可以显著提高化疗效果。评估参与GBM的lncRNA 化疗耐药性，我们确定linc 02454是TMZ治疗后最高度上调的lncRNA之一。 治疗linc 02454的敲低（KD）降低了体外GBM细胞对TMZ的反应活力，而RNA- seq鉴定了CXC-趋化因子受体4型（CXCR 4），是治疗后下调最严重的基因之一。 lncRNA KD. CXCR 4是GBM化学抗性的良好表征的调节剂，目前在多种肿瘤中靶向。 临床试验我们假设TMZ调节linc 02454的表达，linc 02454发挥GBM抗性， TMZ通过CXCR 4的调节。在这个项目中，我们将评估linc 02454对GBM反应的作用 TMZ治疗。在目标1中，我们确定linc 02454是否依赖于CXCR 4表达的增加， 通过上位性和CXCR 4拯救的功能相互作用，以及评估linc 02454是否直接 通过荧光原位杂交调控CXCR 4。在目标2中，我们将评估TMZ如何增加 linc 02454. SA2.1将对DNA基因座（包括linc 02454）的lncRNA甲基化进行整合分析， 甲基化依赖性lncRNA转录在初始切除/复发。SA2.2将评估染色质状态 林可02454在TMZ之前和之后。在目标3中，我们将评估当与TMZ组合时，linc 02454抑制是否 + 放射治疗在体外和体内改善了治疗反应。这些目标将检查lin 02454是否 冥想GBM通过CXCR 4对TMZ的反应以及linc 02454如何受到TMZ暴露的调节。我们还将 测试linc 02454是否是GBM治疗中的潜在治疗靶点。如果成功，这项为期五年的研究将 在TMZ诱导的治疗抗性的背景下提供对lncRNA生物学的更透彻的理解。 与我的导师小组一起，我们制定了一个职业发展计划， GBM lncRNA的转录调控。该项目将通过定期一对一进行密切监督 与初级导师、染色质和转录调控国际专家窦雅丽博士交流 在癌症中。美国国立卫生研究院资助的共同导师，博士Behnam巴迪，神经外科医生专注于GBM生物学，博士。 威廉麦克也有丰富的经验指导神经外科医生，科学家的独立，并已 作为亲密的导师。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，lncRNA的功能和治疗诱导的GBM变化的关键组成部分。

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.