核酸适配体TY04靶向Annexin A2抗多发性骨髓瘤的机制研究

项目名称： 核酸适配体TY04靶向Annexin A2抗多发性骨髓瘤的机制研究

项目编号： No.81470362

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 刘静

作者单位： 中南大学

项目金额： 75万元

中文摘要： 多发性骨髓瘤(MM)发病率随着人口老龄化升高，严重影响健康。基于Cell-SELEX技术筛选到的核酸适配体TY04，为单链DNA分子，长84个碱基，能呈序列特异性与MM细胞稳定结合，抑制MM增殖，阻滞周期于G2/M期，蛋白酶消化实验表明TY04能与膜表面蛋白结合,利用IP技术结合液质联用质谱以及免疫荧光实验，结果提示可定位于细胞表面评分排第一位的膜联蛋白A2(ANXA2)是TY04潜在靶标。本项目提出TY04靶向结合ANXA2，引起ANXA2功能失活，抑制JNK通路活化，影响c-jun活性，导致细胞周期分子紊乱而阻滞于G2/M期，发挥抗MM作用的新设想。本项目将鉴定TY04与ANXA2的结合；明确TY04通过作用ANXA2抑制JNK信号通路激活；在体内外明确TY04通过作用ANXA2发挥抗MM作用。本研究有助于MM治疗新靶标的发现,促进具有自主知识产权的MM核酸类新药研发，具有源头创新性。

中文关键词： C17_骨髓瘤；核酸适配体；膜联蛋白A2；靶标；肿瘤抑制

英文摘要： Multiple myeloma (MM) is the second most common hematological cancer worldwide, accounting for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease for the vast majority of patients, prompting the continued search for additional therapeutic strategies.Aptamers are single-stranded RNA or DNA sequences that can fold into unique three-dimensional (3D) structures, allowing them to form stable and specific complexes with different target molecules of complementary shape. Some aptamers can regulate biological pathways and interfere with disease development through binding to molecular targets involved in pathogenesis. Based on this advantages, aptamers show great potential for therapeutic application. In our previous work, we generated aptamer TY04 through cell-based Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX). We found that TY04 could inhibit the growth of several MM cell lines, which is most sensitive to MM.1S cell line. Flow cytometric analysis showed that TY04 bound to MM.1S cells. Furthermore, we revealed that TY04 induced significant cell cycle arrest at G2/M phase and resulted in the accumulation of binucleated cells. TY04 partly lost its recognition ability when MM.1S cells were briefly treated with trypsin and proteinase K before incubation with TY04, indicating that cell surface membrane proteins must constitute some of the target molecules recognized by TY04. Through IP technology combined with liquid chromatography-mass spectrometry (HLPC-MS) and bioinformatics search, we found that Annexin A2 (ANXA2) was the most potential target molecules of TY04 aptamer. Immunofluorescence staining assay showed TY04 and ANXA2 exist co-localization on MM.1S cell membrane.Thus, we propose a new hypothesis that via targeting ANXA2, TY04 can induce functional inactivation of ANXA2 and inhibition of JNK pathway activation, affect activity of c-jun, and lead to cell cycle arrest at the G2/M phase. This project will study the interaction between TY04 and ANXA2,explore the mechanism of TY04 inhibit the activation of JNK signaling pathway via binding to ANXA2. Explore the effect and mechanism of TY04 anti-MM via targeting ANXA2 in cellular and animals levels. This study will contribute to the development of novel nucleic acid drug for anti-MM.

英文关键词： Multiple myeloma;Aptamer;Annexin A2;Target;Tumor suppression