Lin28A/ROCK2交互作用靶向RAN和HSBP1调控卵巢癌干细胞的功能及分子机制研究

RNA binding protein Lin28A is one of the key factors to reprogram human fibroblast cells into induced pluripotent stem cells (iPS), as well as one of the most important proteins to keep the undifferentiated and pluripotent state of stem cells. Our previous studies showed that Lin28 was high expressed in ovarian cancer tissues, involved in the regulation of biological behaviors in ovarian cancer stem cells, and negatively correlated with the prognosis of patients with ovarian cancer. In addition, Rho-associated coiled-coil containing protein kinase 2, ROCK2, exists in the Lin28A immunoprecipitation complex, and Lin28 binds Ran and HSBP1 mRNA specifically. This project aims to further identify the interaction and binding domain between Lin28A and ROCK2 through fluorescence resonance energy transfer assay and fluorescence co-localization assay, to clarify the molecular mechanism of Lin28A binding to Ran and HSBP1 mRNAs as well as the regulation of the expression of Ran and HSBP1 and their downstream signaling pathways, and to analyze whether the interaction of Lin28A/ROCK2 is required for the regulatory role in the RAN and HSBP1. In the cell, animal and clinical levels, we will study whether Lin28A/ROCK2 interaction controls the expression of RAN and HSBP1 and regulates the ovarian cancer stem cell phenotype, as well as RAN and HSBP1 have the synergistic role to keep the phenotype of cancer stem cells using serum-free suspension culture, cell invasion assay, drug sensitivity assay etc. In addition, the therapeutic effect of ovarian cancer through the destruction of Lin28A/ROCK2 interaction or silencing of RAN and HSBP1 will be studied.The implementation of this project will provide the experimental evidences for elucidating the novel mechanism of Lin28A on the regulation of ovarian cancer stem cells and providing biological novel therapeutic targets for specifically targeting of ovarian cancer stem cells.

RNA结合蛋白Lin28A是重编程获得iPS细胞的关键因子，在维持干细胞多潜能状态中起着重要作用。前期研究发现Lin28A在卵巢癌中高表达，与患者预后呈负相关，能特异结合ROCK2蛋白以及RAN和HSBP1的mRNA。本项目拟通过荧光共振能量转移及荧光共定位等方法鉴定Lin28A/ROCK2交互作用关键结构域；通过报告基因系统查明Lin28A结合RAN和HSBP1的mRNA反应元件；在细胞、动物和临床不同层面，利用无血清悬浮成球培养、细胞侵袭、药物敏感性等实验研究Lin28A是否通过ROCK2靶向RAN和HSBP1表达来调控卵巢癌干细胞的行为，研究RAN和HSBP1表达对卵巢癌干细胞的功能是否具有协同作用；通过消除交互作用或沉默RAN和HSBP1的表达观察对卵巢癌的治疗效果。本项目将为查明Lin28A在卵巢癌干细胞基因表达机制提供新的实验依据，为特异靶向卵巢癌干细胞提供新的生物靶点。

在自然科学基金委的资助下，申请人承担的项目“Lin28A/ROCK2交互作用靶向RAN和HSBP1调控卵巢癌干细胞的功能及分子机制研究”，以Lin28A为切入点，从细胞模型、实验动物及临床样本等不同层面，查明了Lin28A/ROCK2交互作用促进卵巢癌恶性发展的作用机制；发现了Lin28A通过富集RAN和HSBP1的mRNA并上调其蛋白的表达促进卵巢癌细胞干性，增殖和侵袭转移的作用及分子机制；查明了Lin28A,ROCK2,RAN和HSBP1在卵巢癌发生发展中的作用及靶向干预对卵巢癌防治的效果，并且明确了Lin28A,ROCK2,RAN和HSBP1小分子干扰试剂在临床卵巢癌防治中具有潜在价值和应用前景。本项目的研究结果提示Lin28A/ROCK2/RAN/HSBP1高表达可作为卵巢癌患者诊断和预后的标志物，RAN/HSBP1可能成为卵巢癌基因治疗的新靶点。目前相关研究成果发表SCI研究论文24篇，代表性论文发表在Oncogene，Cell Death Dis等国际期刊；授权国家专利6项；参与出版专著1部；培养研究生6名(2人获得国家研究生奖学金)；获得2016湖南省青年科技奖；2017湖南省自然科学二等奖；第十六届湖南医学科技一等奖。