Bmi-1介导CCAT2-miR-200c调控三阴性乳腺癌干细胞和肿瘤发生

Triple negative breast cancer (TNBC) is characterized by aggressiveness, high risk of metastasis and bad prognosis. It is still lacking a specific standard for the clinical treatment. The relapse and metastasis of TNBC are closely related with cancer stem cells, which are under the regulation of non-coding RNAs including miRNA and long non-coding RNA (lncRNA). Our preliminary work made a customized lncRNA panel covering cancer associated lncRNAs. By application to breast cancer cell lines and tissues, we identified a lncRNA, CCAT2, upregulated in cancer cells and cancer stem cells in TNBC. Overexpression of CCAT2 in MDA-MB-231 cells significantly promoted the cell proliferation and cellular migration, and suppressed the expression of miRT-200c. miR-200c was found to be downregulated in TNBC, and regulates breast cancer tumorigenesis by targeting ZEB1 and Bmi-1. Thus, we hypothesized that CCAT2-miR-200c-Bmi-1 pathway regulates cancer stem cell self-renewal and tumor regeneration, CCAT2-miR-200c-ZEB1 pathway regulates cancer cell migration and invasion in TNBC. In this project we will validate the two pathways by analyses on cancer cells, cancer stem cells, clinical samples of TNBC and animal models carrying TNBC tumors. The results will be helpful to reveal the molecular mechanisms through which CCAT2 regulates TNBC, and provide experimental evidence for finding novel target to treat TNBC.

三阴性乳腺癌（TNBC）恶性程度高，转移风险高，预后差，缺乏针对性治疗指南，其恶性程度及复发转移与癌干细胞密切相关，受非编码RNA（miRNA和lncRNA）调控。前期工作制备了癌症相关lncRNA检测panel，进行乳腺癌样本筛查，发现在三阴性乳腺癌细胞及癌干细胞异常高表达的lncRNA CCAT2；在MDA-MB-231细胞过表达CCAT2，能够促进细胞增殖、迁移能力，并特异抑制miR-200c表达。miR-200c在TNBC表达下调，并靶向作用于ZEB1和Bmi-1发挥功能。据此提出TNBC中CCAT2-miR-200c-Bmi-1调控癌干细胞增殖和成瘤，CCAT2-miR-200c-ZEB1调控癌细胞迁移、侵袭的科学假说。本项目将通过TNBC癌细胞、癌干细胞、临床样本及荷瘤动物等研究，验证这两条调控通路，对阐明CCAT2调控TNBC的分子机制，及寻找TNBC治疗新靶点提供实验依据。

三阴性乳腺癌恶性程度高、易转移、缺乏治疗靶点。为了阐明三阴性乳腺癌的发病机制，本项目通过对不同亚型乳腺癌细胞株、临床标本进行lncRNA高通量分析，筛查到一批在三阴性乳腺癌异常高表达的lncRNA, 包括CCAT2。利用三阴性乳腺癌细胞系MDA-MB-231及SRC基因永生化的MCF-10A-Src细胞，分别过表达或者敲低CCAT2证明了CCAT2对三阴性乳腺癌有促进迁移、侵袭、EMT及细胞干性特征的调控功能。利用稳定过表达CCAT2的MDA-MB-231细胞模型，用100个及10000个细胞移植裸鼠制备三阴性乳腺癌动物模型，证明过表达CCAT2可显著促进三阴性乳腺癌的发生及生长。利用基因序列分析及生物信息学工具，发现miR-200c、miR-205、miR-221/222等与CCAT2之间形成非编码基因调控网络，精细调控三阴性乳腺癌干细胞及其复发转移。进一步机制研究揭示了CCAT2-miR-205-NOTCH及CCAT2-OCT4-PG1是介导CCAT2调控三阴性乳腺癌干细胞的两条分子机制。一方面鉴定了miR-205作为CCAT2下游互作基因，能够靶向调控NOTCH2及癌细胞干性。另一方面，发现OCT4的假基因OCT4-PG1作为CCAT2的邻近基因，受CCAT2的正向调控，进而促进癌细胞干性。本研究不仅从长链非编码基因角度阐述了CCAT2对三阴性乳腺癌的调控功能和机制，并为三阴性乳腺癌的治疗提供了一个新的潜在治疗靶点，CCAT2小分子抑制剂具备抑制三阴性乳腺癌干细胞的临床潜力。.本研究相关成果，发表SCI论文7篇，中文期刊论文2篇，授权1项美国发明专利，申请2项中国发明专利。.