多发性骨髓瘤耐药miRNA 标志物的筛选及机制研究

Multiple myeloma (MM) is the tumor characterized by malignant proliferation of plasma cells with complex pathogenesis and widespread drug resistance resulting in low survival rate. It is very important to find diagnostic and prognostic markers. MM drug-resistant miRNA screening is practical in theory and most suitable for obtaining the prognostic markers based on miRNA characteristics and the drug-resistant gene regulatory networks of MM. We are planning to screen and finally confirm the drug-resistant and prognostic miRNA markers by miRNA microarray screening, drug-resistant target gene regulation analysis, real-time PCR with patients' bone marrow and serum samples before and after the cure, combined with the study of the relationship between MM different types and these markers to provide clear drug-resistant diagnostic markers for the clinical treatment of MM. In addition, among the selected markers of our preliminary studies, miR-202, which is newly proved to be the miRNA that can regulate B-cell activating factor, needs our study to clarify its role in MM drug-resistant mechanism. We will also confirm the role of miR-202 in the final common markers. This study will obtain some clear miRNAs as MM drug-resistant and prognostic biomarkers, at the same time, we can also clarify some new MM drug-resistant regulation mechanisms to provide targets for novel drug development.

多发性骨髓瘤（MM）是浆细胞恶性增生肿瘤，发病机制复杂，普遍耐药导致生存率低是该病的特点。因此，确认耐药诊断和预后判断的标志物很重要。根据该病耐药基因调节网络和miRNA的特点，筛选耐药miRNA是理论可行且最适该病预后判断标志物获得的窗口。本研究拟选择用药前后MM患者骨髓及血清样本，通过miRNA芯片筛选、耐药靶基因调节分析、荧光定量PCR验证来进行耐药及预后诊断miRNA标志物的筛选和最终确认，并研究其与MM不同发病类型的关系，为MM临床治疗提供明确的耐药诊断标志物。另外，在前期研究备选标志物中，miR-202是我们新证明的可调节B细胞活化因子的miRNA，其在MM耐药的机制有待本研究阐明。在最终标志物的公共性确认中，我们也将对miR-202进行认证。本课题的开展将获得一些明确的miRNA作为MM耐药及预后判断的标志物；同时可探明MM新的耐药调节机制，为新药开发提供靶点。

多发性骨髓瘤（Multiple myeloma，MM）是浆细胞异常增生的B细胞恶性克隆性疾病。研究表明，miRNA在肿瘤中常发挥癌基因或抑癌基因的作用，参与肿瘤的发生，发展及耐药等行为。肿瘤细胞可释放miRNA进入外周血循环。肿瘤病人外周血循环中miRNA表达显著异常，有组织或细胞特异性，能在血清或血浆中稳定存在。我们拟检测MM患者用药前后血清及细胞中miRNA基因表达水平，以期得到特异性的用于MM耐药诊断及预后判断的miRNA标志物。我们在本项目的资助下，首先利用基因芯片筛选MM用药前后变化的miRNA分子，发现上调的miRNA分子11个（miR-130、miR-199、miR-let7等），下调的miRNA分子105个（miR-29b、miR-193、miR-148a、miR-138等）。然后建立SYBR GreenⅠ荧光定量PCR检测血清miR-202等miRNA分子的方法，并对方法学进行评价。研究发现miR-202、miR-148a、miR-193、miR-4449等分子在MM患者外周血中呈现差异表达。另外，这些分子与β2M、轻链λ、轻链κ浓度等临床指标存在相关性。进一步对miRNA分子在MM中的功能研究显示，miR-19b/20a miR-202、miR-148a、miR-193负调控其靶基因促进MM细胞增殖、迁移，抑制细胞凋亡。机制研究发现，这些miRNA分子可激活NF-κB、JNK/SAPK等信号通路促进MM细胞生长增殖、迁移并且抑制凋亡，从而促进病程的进展。另外，复发/进展期MM患者中miR-335启动子区甲基化程度高于初诊患者，miR-335的甲基化与MM细胞的迁移能力密切相关。本研究有望为临床耐药及具有预后判断价值的miRNA分子的检测、肿瘤的基因诊断与肿瘤发病及耐药机制的研究提供新的策略和思路，为开发药物提供新的靶点。