遗传印迹长非编码RNA-IRAIN在乳腺癌干细胞中的抑癌机理研究

The cancer stem cell (CSC) hypothesis argues that a small minority of cells in a heterogeneous tumor population drives tumor growth. CSCs play an important role in tumor initiation, progression, and metastases. The insulin-like growth factor-I receptor (IGF1R) is overexpressed in breast cancer. Through the activation of the PI3K/AKT and MAPK signal cascades, IGF1R plays an important role in regulating the self-renewal of CSCs and promoting cell proliferation and resistance to chemotherapeutic agents, radiation and targeted therapies using tamoxifen and herceptin in breast CSCs. IGF1R is a well-established drug target. Drugs targeting this pathway are currently being tested in clinical trials...To study the dysregulation of this pathway in cancer, we used a R3C (RNA-guided Chromatin Conformation Capture) approach to discover a novel intragenic long noncoding RNA (lncRNA) in the IGF1R locus, which we have called the “IGF1R antisense imprinted noncoding RNA” (IRAIN). In breast cancer, this lncRNA is expressed in a monoallelic manner in an antisense orientation. Most interestingly, IRAIN binds to the chromatin DNAs at the enhancer and promoter of IGF1R, reversely regulating it expression, suggesting its tumor suppressor role in breast CSCs. We propose to completely characterize the mechanism underlying tumor suppressor activity of IRAIN lncRNA in breast CSCs. We will address the following challenges:.1..Does IRAIN noncoding RNA participate in the regulation of the self-renewal of breast CSCs through the IGF1R pathway? We assess the biological role of this lncRNA by deleting it from breast CSCs using CRISPR Cas9 RNA-guided genome editing approach.2..Will ectopically expressed IRAIN noncoding RNA reduce the tumorigenicity of breast cancer stem cells? .3..Does IRAIN lncRNA function as a tumor suppressor by competing oncogenic miRNAs? Using oligo-affinity precipitation (OAP) assay, we will examine how this lncNRA competes with oncogenic miRNAs to suppress cancer growth..4..What are genomic target sites of IRAIN lncRNA. We will use a “reverse transcription-associated trap” (RAT) assay to identify chromatin DNAs, wherer this lncRNA binds and recruits chromatin factor EZH2 and CTCF in gene regulation. .5..Is IRAIN noncoding RNA a reliable prognostic marker in breast cancer patients? We will utilize a tumor bank of breast cancer specimens to learn if IRAIN abundance correlates with breast cancer outcomes...The full characterization of this intragenic lncRNA will provide important information regarding the physiology and pathophysiology of IGF1R in breast cancer stem cells, and may suggest a new target for anti-IGF1R therapeutics. Moreover, IRAIN abundance may represent a new biomarker for prognosis or therapeutic decision-making in patients with breast cancer..

肿瘤干细胞具有高度增殖活性和自我更新能力，参与肿瘤的发生、进展、复发、转移及化放疗耐药。类胰岛素生长因子I受体（IGF1R）在乳腺癌中高表达，通过自身磷酸化激活PI3K、MAPK信号通路，调控肿瘤干细胞的自我更新，是公认的肿瘤靶基因之一。本课题组采用R3C方法首次发现与IGF1R反义、且呈父源印记表达的长链非编码RNA (IRAIN)基因。IRAIN表达与肿瘤恶性度呈负相关，并通过负向调控IGF1R，影响肿瘤生长和转移。本课题将采用CRISPR RNA导向基因修饰技术敲除IRAIN启动子及用病毒过表达非编码RNA的方法，明确IRAIN在乳腺癌干细胞的抑癌作用；通过检测其竞争内源靶RNA（ceRNA）、结合靶向基因染色质DNA、募集染色质因子EZH2等，探索IRAIN抑癌的表观遗传学分子机制；分析其表达与乳腺癌预后的关系，揭示其在乳腺癌发病中的作用，为临床治疗及新靶向药物开发提供理论依据。

类胰岛素生长因子I受体（IGF1R）是恶性肿瘤的热门分子靶点，但其调控机制不清。我们在IGF1R区域发现一条全新的呈单等位基因印记表达、且与启动子重叠的反义lncRNA，命名为IRAIN。乳腺癌中IRAIN表达与肿瘤恶性度呈负相关，IRAIN与IGF1R相互作用调控乳腺癌生物学特性的分子机制是本项目的研究重点。.我们结合临床样本阐明IRAIN在正常乳腺组织和乳腺癌各分子亚型中的表达水平及与预后的相关性；首次发现IRAIN在肿瘤中存在异常的“等位基因印记转换” (allelic switch) 表达，即乳腺癌患者的正常-癌组织、原发-转移灶中，呈现父母不同来源的单等位基因表达；发生IRAIN印记转换的肿瘤存在广泛的DNA去甲基化。建立全新的ALIC(antisense lncRNA in situ cis-competition)靶向技术，实现IRAIN原位(in situ)反义lncRNA过表达，达到顺式(in cis)原位竞争抑制IGF1R，证明IRAIN对IGF1R的顺式调控可抑制乳腺癌的肿瘤潜能性。采用核内原位逆转录捕获（RAT）测序技术，绘制IRAIN全基因组靶点DNA互作网络图谱；发现IRAIN通过反式(in trans)机制结合并调控多种肿瘤相关基因；发现IRAIN抑制肿瘤转移抑制基因NM23，通过表观修饰阻断乳腺癌生长和转移。本研究为乳腺癌个体化靶向治疗及预后提供了理论基础和实验依据。 .同时探讨非编码RNA在恶性肿瘤中的生物学作用和机制，阐明MALAT1在乳腺癌中的靶分子EEF1A1；发现miR-483-5p与IGF2启动子结合而促进其表达；发现新的环状RNA FECR1及其对乳腺癌的表观调控；建立Cas9-EpiS靶基因甲基化技术，通过诱导启动子甲基化抑制肝癌细胞颗粒蛋白的表达；建立CRIST-seq技术，鉴定与Oct4和Sox2启动子结合的干细胞调控lncRNA图谱。本课题从多角度多层面对非编码RNA与肿瘤发生发展的关系做了深入研究。.在项目资助下共发表了28篇标注文章，有望为后续研究提供新的线索。获得2项发明专利，培养了20多名博士生和青年科研人员。