BUB1B调控多发性骨髓瘤细胞增殖和耐药作用及机制研究

Multiple myeloma is an incurable plasma cell malignancy. Although the development of MM treatment strategies have been advanced in recent years, most of MM patients relapsed, became drug-resistant and died of MM eventually. Therefore, seeking for novel target on MM proliferation and drug-resistance is one of the most important mission for MM research. Through microarray analysis, we discovered that BUB1B increased in MM samples compared to normal plasma control cells. Meanwhile, BUB1B expression enriched in relapse and sequential MM samples relative to newly-diagnosed samples. Importantly, patients with high expression of BUB1B were associated with poor survival. Preliminary study showed increased BUB1B induced MM proliferation and drug-resistance. In this study, we plan to utilize MM primary samples, MM cell lines, 5TGM1 and BCL2/IL6 transgenic mouse models and employ PET/CT image, RNA sequencing, proteomics, immunohistochemistry staining, CRISPR technics to explore the chromosomal instability, drug-resistant, clonogenic characteristics of BUB1B in vitro and in vivo, verify its role on MM diagnosis and pharmacological target and clarify the mechanism. Finally, we intent to screen the targeting drug on BUB1B based on compound library of natural medicine. Summarily, our study aims to provide strong theoretical and clinical support for MM diagnosis and therapy.

多发性骨髓瘤（MM）是不可治愈的浆细胞恶性肿瘤，绝大多数患者复发并出现耐药。因此，开发新的治疗靶标是目前MM研究重点。运用基因芯片，本人发现BUBIB基因在MM细胞中表达高于正常浆细胞，在复发、序贯样本中表达高于初诊样本，高表达BUB1B患者生存率显著降低。初步研究显示BUB1B引发MM细胞增殖与耐药。本项目拟在MM患者样本中检测BUB1B与染色体不稳定、增殖与耐药的相关性；运用CRISPR技术调控BUB1B在MM细胞中表达，研究其体外引发MM染色体不稳定、耐药及增殖等作用；通过5TGM1、BCL2/IL6转基因小鼠模型结合过继性B细胞技术在体内确定BUB1B上述功能；应用免疫共沉淀、点突变、蛋白组学、RNA测序等技术阐明其作用机制，验证其作为MM标志物及药物靶点可行性。最后，利用天然化合物库筛选BUB1B抑制剂, 并研究其药理活性。本课题拟为MM治疗提供新的诊断工具、药物靶点及治疗手段。

染色体不稳定是导致多发性骨髓瘤（MM）恶性增殖、复发耐药的重要原因之一。 申请人研究发现：BUB1B基因在MM复发样本中表达明显高于初诊样本，高表达BUB1B患者生存率显著降低。在体外细胞实验和骨髓瘤小鼠模型中，过表达BUB1B促进细胞增殖，诱导MM细胞耐药。机制研究揭示，BUB1B蛋白含有激酶催化活性中心，过表达BUB1B主要通过磷酸化CEP170的ser1260位点从而引发染色体不稳定促进MM细胞增殖且导致MM细胞耐药。另外，本课题从骨髓微环境角度出发，发现环状RNA circBUB1B（has_circ_0034566）在MM病人血液、肿瘤、MM细胞中高表达，其编码的蛋白circBUB1B_544aa，含有BUB1B激酶催化活性中心，通过磷酸化CEP170从而引发染色体不稳定诱导MM细胞耐药。此外，MM细胞可以向骨髓肿瘤微环境中分泌circBUB1B从而影响邻近的细胞。同时敲低BUB1B和circBUB1B_544aa在体内外抑制了MM的增殖和耐药，表明BUB1B和circBUB1B作为药物靶标的可行性。本项目拟为MM诊断与治疗提供新的手段。