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

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。

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..