NLS-RARα作为APL早期诊断标志物的研究

Promyelocytic leukemia-retinoic acid receptor α (PML-RARα) is the main pathogenic factor for the development of acute promyelocytic leukemia (APL). PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specifc serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. Two mutant proteins named PML (NLS-) and NLS-RARα were produced. The present study was designed to analyze the role of the NLS in mediating RARα transport into the nucleus and to evaluate the value of measuring NLS-RARα in the early diagnosis of APL. The localization of NLS-RARα and the molecular mechanism of its transportation to the nucleus are examined by the indirect immuninofluorescence labeled Laser Co-focus light microscopy and co-immunoprecipitation (CO-IP). The lentivirus containing the NLS-RARα gene is transfected into NB4 cells to evaluate its effects on leukemogenesis induced by NB4. The techniques of qPCR, Western Blot and CO-IP are adopted to explore the mechanism of NLS-RARα in inducing APL. Whether the NLS-RARα protein could be a novel diagnostic biomarker for APL would be investigated by preparing its monoclonal antibody and then detecting its expression and localization in early APL cases. This study lays the experiment and theory foundation for investigating the mechanism driving APL, depicting a new pathogenic signal pathway of PML-RARα, and discovering new targets and methods for the diagnosis and treatment of APL.

PML-RARα融合蛋白是APL发生的关键因素，早期髓细胞中高表达的中性粒细胞弹性蛋白酶(NE)可将PML-RARα裂解成52KDa的PML(NLS-)和61KDa的NLS-RARα两种变异蛋白。NLS-RARα由于带上了核定位信号（NLS）而发生了定位与功能的改变，在APL的发生中具有潜在的重要作用。目前除了我们的研究外，国内外尚无相关的研究与报道。本课题组拟在前期研究基础上，通过间接免疫荧光标记的激光共聚焦显微成像与免疫共沉淀(CO-IP)等技术研究NLS-RARα的定位及其入核机制；通过构建稳定转染NLS-RARα的NB4细胞株研究其对NB4细胞致小鼠白血病成瘤能力的影响；通过qPCR、Western Blot、CO-IP等方法研究NLS-RARα促白血病的机制；进一步通过制备NLS-RARα的特异性抗体，在早期APL病例中研究其表达与定位，探讨其能否作为APL早期诊断标志物。

PML-RARα融合蛋白是APL发生的关键因素，NE可将PML-RARα裂解成PML（NLS-）和NLS-RARα两种变异蛋白。本项目主要围绕NLS-RARα开展研究。主要研究内容包括：1）NLS-RARα蛋白的表达及细胞定位研究；2）NLS-RARα的生物学功能及其致病机制研究；3）NLS-RARα入核过程研究；4）NLS-RARα在APL发生中的诊疗新靶点的探索。研究结果表明：1）新型变异蛋白NLS-RARα是PML-RARα的切割产物，相比于野生型RARα蛋白，其在APL细胞中的表达和定位有显著差异。2）NLS-RARα可通过维甲酸信号通路抑制细胞分化和调控细胞周期，进而促进APL的发生发展。（3）NLS-RARα促进白血病小鼠APL样症状的发生和进展，缩短小鼠生存时间。4）NLS-RARα主要定位于细胞核，其依赖于RARα自带的NLS入核并参与调控细胞分化，其入核机制主要是与KPNA2、KPNB1形成三联复合物。5）NLS-RARα是APL早期生物标志物，基于NLS-RARα特异性单抗为核心研发的相关技术产品可以用于APL的早期诊断。本项目的研究成果为深入揭示APL发生的分子机制和绘制新的致病信号途径奠定了实验与理论基础，为探寻新的APL诊疗靶点提供了新的思路。通过本项目实施，共发表核心期刊论文24篇，其中SCI论文16篇，申请国家发明专利1项，共培养博士研究生6名、硕士研究生17名。