核酸适配体TY04靶向Annexin A2抗多发性骨髓瘤的机制研究

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.

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。

多发性骨髓瘤（Multiple myeloma，MM）是浆细胞来源的血液系统的恶性肿瘤。虽然其治疗已取得很大进展，但由于其易复发，仍是一种不可治愈的血液肿瘤，因此需要开发诊断和治疗MM的新方法。膜联蛋白A2（Annexin A2，ANXA2）是Annexins超家族中A亚家族的重要成员，MM患者高表达ANXA2，且高水平ANXA2与患者总存活率和无病生存率负相关，提示ANXA2是MM潜在的诊断和治疗靶标。核酸适体通过折叠形成特定的三维结构与靶标分子结合，具有高亲和力、高特异性、低免疫原性和生物毒性、组织渗透力强、易于合成和标记等特点，核酸适体在临床诊断与治疗方面展示出较大应用潜力。.本课题拟针对ANXA2蛋白筛选特异性核酸适体，首先我们构建了pET-42a-ANXA2的原核表达载体，通过IPTG诱导表达，纯化后获得GST-ANXA2重组融合蛋白。然后利用protein-SELEX技术，使用GST-ANXA2重组融合蛋白作为筛选靶标，GST蛋白作为负性筛选；以包含有80个碱基的随机单链DNA寡核酸库作为初始库，通过逐步减少正筛孵育时间，增加洗涤次数和负筛孵育时间等增加筛选压力，经过九轮筛选后将第九轮筛选产物通过高通量测序。通过测序和酶联寡核苷酸分析，wh6核酸适体对ANXA2蛋白的亲和力最高，因此wh6被作为本课题后续研究对象。.通过提取MM细胞的膜蛋白并与生物素标记的wh6核酸适体孵育，Aptamer-pulldown结果也证明wh6能与细胞中ANXA2蛋白结合。通过酶联寡核苷酸分析wh6与ANXA2蛋白的结合常数为8.75±1.26 nM。通过shRNA慢病毒敲低MM细胞中的ANXA2蛋白，wh6与MM细胞结合能力减弱。建立SCID免疫缺陷小鼠异体移植MM细胞成瘤模型，结果显示wh6在体内与ANXA2蛋白高表达的MM细胞具有强靶向结合能力。多发性骨髓瘤患者样本结合实验显示，与DNA-lib相比，wh6能与多发性骨髓瘤患者骨髓细胞中CD138阳性细胞显著结合。同时，wh6可以抑制由ANXA2诱导的RPMI-8226和MM.1S细胞的生长，并能阻断MM细胞对ANXA2粘附。.综上，本课题筛选到靶向ANXA2蛋白的核酸适体wh6可作为多发性骨髓瘤诊断和治疗的候选工具。