建立活性可控的基因编辑系统并靶向特异性超级增强子的三阴性乳腺癌治疗实验研究

Breast cancer is the most common malignancy in Chinese women. Among them, triple-negative breast cancer (TNBC) seriously threatens the health of Chinese women because of the lack of therapeutic targets and effective treatment methods, resulting in a short survival period of patient. Therefore, it is of great value to find new therapeutic targets for TNBC and develop safe and controllable gene therapy approaches. Recently，a research published on Nature reported that therapeutic strategies targeting tumor-specific super-enhancers were effective in the treatment of ependymoma. Inspired by this, this project is intended to develop a safe and effective tumor gene therapy method used by tumor-specific super-enhancers targeting in TNBC. This project team aims to obtain innovative and therapeutic strategies targeting to the specific super-enhancer of MDA-MB-231 cell line which is a TNBC cell line through a series of bioinformatics analysis and prediction based on the ChiP-seq and RNA-seq Data, STARR-seq high-throughput screening and CRISPR site screening methods. Initially, the mechanism of relevant targets pathway is clarified. Simultaneously, a genome editing system of which the activity can be regulated, is expected to develop, so that gene therapy targeted to TNBC specific super-enhancers could be more safe and controllable. Up to now, all the study result of the project were become as expected. It was believed that the finding of this project will provide new therapeutic targets, ideas and methods for the treatment of TNBC.

乳腺癌为中国女性发病率最高的恶性肿瘤，其中三阴性乳腺癌由于缺乏明确的治疗靶点，导致现有治疗手段单一，病人生存期短。故发现新的三阴性乳腺癌治疗靶点及开发靶向新靶点的安全可控的基因治疗手段意义重大。近期Nature报道，以肿瘤特异性超级增强子为靶点的治疗策略在室管膜瘤的治疗实验中效果显著。故本项目拟开发一种靶向三阴性乳腺癌肿瘤特异性增强子的安全有效的基因治疗方法。本项目拟通过基于ChiP-seq和表达谱数据的系统性生物信息学分析预测，STARR-seq高通量筛选及CRISPR位点筛选等方法，创新性的获得针对三阴性乳腺癌MDA-MB-231细胞系的特异性超级增强子治疗靶点，并初步阐明相关靶点的治疗机理。同时开发出一种活力受到四环素严密调控的基因组编辑系统，使靶向超级增强子的基因治疗安全可控。本项目的已完成的研究结果均符合预期，其最终结果将为三阴性乳腺癌提供新的治疗靶点，思路和方法。

三阴性乳腺癌与其它乳腺癌类型相比预后较差，具有更高的复发风险和更短的存活时间。因此，找到一种新的三阴性乳腺癌的治疗分子靶点是非常必要的。而超级增强子与肿瘤的发生发展密切相关。本研究通过对三阴性乳腺癌组织和细胞的数据分析，预测出12,164个特异性超级增强子的核心调控元件序列。结合本研究开发的独特的基于慢病毒的Lenti-STARR-Seq高通量分子生物学技术筛选方法，获得了3个三阴性乳腺癌特异性超级增强子序列及其靶基因SIAH2。通过CRISPRi的定点干预，确定了其中2个靶点序列rs16862837_sgRNA1与rs2018246_sgRNA3可以有效下调SIAH2，并能有效抑制三阴性乳腺癌细胞的体外增殖。同时发现了ER阳性细胞中3个ESR1基因的超级增强子靶点，及两个潜在的新的致癌基因PRGRIP1L与ZMIZ1。本研究的成果有助于解决三阴性乳腺癌缺乏治疗靶点的难题，并为筛选缺乏治疗靶点的疾病的靶点开发提供了新的思路。同时，本研究开发出一种活力受到四环素严密调控的基因组编辑系统，使靶向超级增强子的基因治疗安全可控。