类胰岛素生长因子I受体反义长链非编码RNA作为抑癌基因在乳腺癌的印记表达及表观遗传学调控

The insulin-like growth factor (IGF) pathway plays a critical role in initiation and progression of breast cancer. Overexpression of IGF1R activates the PI3-K and MAPK signal cascades, resulting in cell proliferation, pro-apoptosis, and the resistance to chemotherapeutic agents, radiation, and targeted therapies using tamoxifen and herceptin. A variety of drugs targeting this pathway are currently being tested in clinical trials. However, the mechanisms underlying IGF1R dysregulation in tumors remain enigma. To study the dysregulation of this pathway in cancer, we have used a R3C (RNA-guided Chromatin Conformation Capture) method recently developed in our lab to identify the presence of a novel long noncoding RNA (lncRNA) in the IGF1R locus. In breast cancer, this lncRNA is expressed in a monoallelic manner in an antisense orientation. It is thus named as IRAIN (IGF I receptor antisense imprinted noncoding RNA). Most interestingly, IRAIN is expressed reversely with IGF1R, with high abundance in in situ tumors but low or undetectable in metastatic tumors, suggesting its tumor suppressor role in regulating IGF1R expression in tumors. This project is proposed to address several challenging issues: 1) Is mono-allelic expression of the lncRNA altered in breast cancers? 2) What is the mechanism for the monoallelic transcription of IRAIN? 3) How does this antisense lncRNA regulate IGF1R expression? 4) What role does aberrant allelic expression of IRAIN play in tumor development? and 5) Does knockdown of this lncRNA affect the growth and invasiveness of breast cancer cells? Specifically, we will characterize the allelic expression of IRAIN and epigenetic regulation in breast cancers. We will examine if aberrant expression of IRAIN is associated with IGF1R dysregulation in tumors. We will also determine the role of IRAIN in oncogenesis using targeted methylation silencing and shRNA knockdown approaches. The data obtained from this study will shed light on the molecular mechanisms for dysregulation of the IGF pathway in breast cancer. In addition, this project will provide a mechanistic rationale for designing antitumor drugs that target the overexpressed IGF1R in breast cancer by harnessing the lncRNA epigenetic machinery.

类胰岛素生长因子I受体（IGF1R）在乳腺癌及多种肿瘤中高表达，通过自身磷酸化激活PI3K及MAPK信号通路，促进肿瘤生长，其表达与肿瘤的生长转移、放化疗治疗抗性密切相关，但其高表达的具体机制尚不清楚。本课题采用R3C方法首次在IGF1R基因区发现一条长链非编码RNA (lncRNA)，该lncRNA与IGF1R呈反义表达，在正常组织中印记表达，命名为IRAIN (IGFI receptor antisense imprinted noncoding RNA)。IRAIN在乳腺原位癌中表达高于非原位癌，与IGF1R mRNA表达呈相反趋势，提示IRAIN可能作为抑癌基因，负向调控IGF1R表达，影响肿瘤生长和转移。本课题将分析IRAIN结构及功能，明确其在肿瘤中的印记表达变化，探索IRAIN调控IGF1R表达的机制，揭示IRAIN在乳腺癌发病中的作用，为临床治疗及及新药研究提供理论依据。

类胰岛素生长因子I受体（IGF1R）在乳腺癌及多种肿瘤中高表达，与肿瘤的生长转移、放化疗治疗抗性密切相关，但其高表达的具体机制尚不清楚。长链非编码RNA（lncRNA）能通过表观遗传机制调控肿瘤细胞基因表达，参与肿瘤的发生及转移，但目前尚无任何关于调控IGF1R表达的lncRNA的报道。.课题申请人在国际上首次发现一条全新的长链非编码RNA，将其命名为IRAIN。课题组在乳腺癌中进行了IRAIN全长图谱绘制，确定了IRAIN表达方向、启动子区域及核内外定位。 在乳腺癌细胞系中发现 IRAIN 表达低于 IGF1R，呈反向趋势，在预后相对差的 TNB 和 HER2+乳腺癌病人中，IRAIN 表达明显下降。.在人体正常乳腺、胃、心脏、子宫等组织中，在乳腺癌细胞系、乳腺癌病人癌组织中IRAIN 呈单等位基因印迹表达。在正常人外周血及乳腺癌病人外周血中均为单等位基因表达，且在部分病人中“A”等位基因偏倚表达。进一步分析还发现，有部分病人存在 IRAIN“印迹转换”现象，即外周血和癌组织之间 IRAIN 表达不同的等位基因，可能作为早期筛查及预后随访标志，机制分析不除外与表观遗传学改变相关。.功能及机制方面，课题组在反式水平通过慢病毒过表达 IRAIN 后发现其抑制乳腺癌细胞系迁移，但对 IGF1R 均无明显影响。故进一步采用CRISPR-CAS9 基因编辑方法，从顺式水平上增强 IRAIN 表达，结果发现IRAIN明显抑制 IGF1R ，抑制乳腺癌增殖、转移。说明 IRAIN 可能在顺式水平上抑制IGF1R 表达及功能，发挥抑癌作用。.进一步通过逆转录相关捕获法（RAT）、RNA-染色质构象捕获方法（R-3C）、染色质构象捕获法（3C）明确了IRAIN作用机制，即其与 IGF1R 连接，介导IGF1R启动子与增强子之间的内环形成。.课题组在研究内容上延伸，通过NRAT（核 lncRNA 逆转录捕获-测序分析）及生物学分析方法研究 IRAIN 下游作用基因网络及可能相关的基因，初步发现NM23可能与IRAIN作用相关。.本课题结果初步揭示IRAIN在乳腺癌发病中的作用及机制，为临床治疗及及新药研究提供了坚实的理论依据。