低氧-HIF1α调控的LncRNA促三阴性乳腺癌干细胞干性维持与肿瘤转移的分子机制研究

Hypoxia is a major effector to tumor malignancy and development in solid tumor microenvironment. Cancer stem cells (CSC) play a critical role on tumor metastasis, drug resistance and recurrence. Our previous studies using HIFa-knocked down triple-negative breast cancer (TNBC) showed a HIF1a-dependant hypoxic features of cell invasion, cancer stem cell enrichment, gene and LncRNA expressions. DNA sequences and bio-informatics analysis indicated that three new LncRNAs may coordinately regulate Hippo signaling to involve in CSC stemness maintenance and tumor cell invasion. Interfering one of LncRNAs led to decreased stemness of BCSC and invasive ability. Thus, it raises an intriguing scientific question that these HIF1a-dependant novel LncRNAs may play roles in stemness maintenance and cell invasion of TNBC. To further verify this hypotheses, we try to study the mechanism of these LncRNAs in regulation of mammospheres formation, stemness maintenance, drug resistance of TNBC-CSC and tumor cell invasion and metastasis in vitro and in vivo using siRNA, RIP-Seq, and LC-MS/MS to find the downstream targets, interacting proteins or related signaling associated with Hippo. The results of this program will have critical scientific and clinical values for understanding tumor metastasis and recurrence of TNBC.

低氧是影响实体肿瘤恶性进展的重要体内环境因子。肿瘤干细胞（CSC）是导致肿瘤转移、耐药、复发的根本。前期研究发现低氧下HIFα敲除的三阴性乳腺癌（TNBC）体现出HIF1α依赖的侵袭、CSC富集及基因、LncRNA表达特性；生物信息分析测序结果揭示3个新LncRNA协同调控Hippo信号可能参与CSC干性维持、转移。干预其中一个LncRNA表达，降低CSC干性与侵袭。推测这些HIF1α依赖的LncRNA调控TNBC-CSC干性与转移。该项目拟进一步采用siRNA干扰、RIP-Seq、LC-MS/MS等手段，研究这些LncRNA对mammospheres形成、干性维持、小鼠致瘤，癌细胞侵袭转移、药物耐受变化；寻找LncRNA通过靶基因或互作蛋白，影响下游Hippo及相关信号，调控TNBC-CSC干性维持、转移复发分子生物机制。研究结果将助于加深了解低氧对TNBC转移复发具重要理论与临床价值。

经课题组全体人员共同努力，圆满完成了各项研究内容，取得如下丰硕的成果：1.受低氧特异诱导的新lncRNA KB-1980E6.3调控乳腺肿瘤干细胞干性。证实lncRNA KB-1980E6.3通过招募IGF2BP1，与c-Myc互作，维持c-Myc mRNA的m6A高甲基化，保证其稳定性和高表达，在低氧诱导的BCSC中发挥着核心作用。lncRNA KB-1980E6.3是评估乳腺肿瘤患者预后不良的良好指标。2.揭示lnc408是EMT化乳腺癌干细胞中特异表达的、维持BCSC干性的一个重要的ncRNA。Lnc408募集转录因子SP3到CBY1启动子区，抑制CBY1表达；Lnc408介导的CBY1低表达阻碍14-3-3/β-catenin/CBY1复合体形成，维持β-catenin的核内定位，调控BCSC相关基因表达。此外，lnc408还参与乳腺肿瘤转移。lnc408作为miR-654-5p的海绵样吸附体，抑制miR-654-5p，上调LIMK1表达。高表达的LIMK1磷酸化p-cofilin和CREB，影响F-actin稳定性，以及MMP2、ITGB1、COL1A1表达，促乳腺肿瘤侵袭转移。3.发现低氧诱导的SLC6A8受p65/NF-kB调控， 转运与富集肌酸，特异激活AKT-ERK信号，降低线粒体呼吸链活性与ROS产生，促进低氧乳腺癌细胞生存。4.缺氧诱导因子1α(HIF-1α)转录表达的新型circSTT3A调控BCSC干性。circSTT3A介导并促进HSP70与PGK1结合成三元聚合体，抑制PGK1泛素化降解。PGK1催化1,3-二磷酸甘油酸形成3-磷酸甘油酸酯，产生丝氨酸，参与BCSC干性维持。5.鉴定并证实低氧特异诱导的lncPCAT6，选择性地介导BCSC干性相关m6A甲基化修饰的mRNA出核，参与干性维持。揭示lncPCAT6结合ISG15，使ISG15与m6A阅读器蛋白hnRNPA2B1互作解离，消除ISG15对hnRNPA2B1类泛素化降解，维持hnRNPA2B1蛋白稳定。增高的hnRNPA2B1特异识别m6A修饰的、CSC干性相关mRNA（如fgfr1）核输出，发挥促CSC干性特征。阿霉素联合STM2457（m6A抑制剂）可有效消除BCSCs和肿瘤发生。.发表高水平SCI收录论文4篇（1篇IF>10.0）；在审2篇；培养博/硕士生各3名。