启动子甲基化沉默miRNA-33b靶向调控P53/MDM2信号通路参与多发性骨髓瘤发病的机制研究

Cytogenetic abnormalities are the hallmark of multiple myeloma (MM) and commonly used as the clinical predictors for determining the stage of disease and providing the guidance for therapeutic strategies. Risk-stratification system based on genetic indicators has been established and recommended by mayo clinic and International Myeloma Working Group (IMWG) in recent years. However, current prognostic factors including del(p53), t(4;14) and amp(CKS1B) can not completely explain the heterogeneity in this disease. So, identifying some novel prognostic markers to better risk stratify patients in the management of myeloma and investigating their roles in myeloma pathogenesis are becoming important issues. Our previous results showed miRNA-33b might be a novel prognostic factor according to the results of serum microRNA microarray. To further ascertain the expression of miRNA-33b in different stages of MM patients, we detected the expression of miRNA-33b in 21 newly diagnosed, 8 relapsed, 8 complete remission (CR) MM patients and 11 normal samples. Our results showed the expression of miRNA-33b was significantly lower in newly diagnosed and relapsed MM patients than CR MM patients and normal samples, and the patients with low expression of miRNA-33b had shorter overall survival (OS) time than control patients. Further studies showed that there was obviously hypermethylation in the promoter region of miRNA-33b in myeloma cells. In addition, transient overexpression of miRNA-33b in U266 and RPMI 8226 cell lines could downregulate the expression of MDM2 protein and upregulate the expression of P53 protein. So, we hypothesize the low expression of miRNA-33b via methylation of promoter CPG region contributes the pathogenesis of myeloma by targeting the p53/MDM2 signal pathway which controls the proliferation and apoptosis. In this project, we will comprehensively investigate the mechanism that miRNA-33b participates in the pathogenesis of MM in vitro and vivo and evaluate the prognostic value of miRNA-33b in MM patients. Taken together, this project will provide the clinical and experimental evidences for miRNA-33b as a novel prognostic factor and valuable therapeutic target.

细胞分子遗传学异常是影响MM预后的最重要因素，补充新的分子预后指标，建立完善的危险度分层体系和个体化治疗策略是MM未来发展的趋势。申请人根据前期血清miRNA芯片结果，选择miRNA-33b在MM患者和正常浆细胞中进行验证，结果miRNA-33b在初治和复发患者中表达明显降低，miRNA-33b低表达的患者总生存时间明显缩短，进一步研究发现，MM细胞miRNA-33b启动子区域甲基化程度明显高于正常人，且miRNA-33b表达与MDM2负相关，与P53蛋白表达呈正相关。据此推测miRNA-33b启动子区域甲基化致其低表达，并通过靶向MDM2进而调节P53蛋白参与MM发病。本课题拟对其具体作用机制进行研究，验证MDM2为其直接作用的靶基因，初步探索去甲基化药物5-Aza 靶向miRNA-33b对MM的治疗作用，为探索以miRNA-33b及其靶基因为新靶点的生物治疗提供可靠的理论和实验依据。

背景：研究非编码RNA在MM中的发病机制，寻找新的MM分子预后指标，建立完善的危险度分层体系和个体化治疗策略。.主要研究内容：.阐明MM miRNA-33b启动子区域存在甲基化是导致其低表达的原因；体内外实验证明miRNA-33b影响MM细胞的增殖、凋亡、细胞周期和克隆形成能力明确其具体调节的细胞周期和凋亡蛋白；体外实验证明使用去甲基化药物治疗能使MM细胞系和原代细胞的增殖和克隆形成能力减低，促进凋亡；检测临床患者miRNA-33b表达并结合临床资料分析，为miRNA-33b作为有价值的预后分子标记提供的临床证据。.研究结果和关键数据：.1..miRNA-33b在18例健康供者，58例初治MM，11例复发MM，12例缓解MM中检测其表达水平，初治和复发MM miRNA-33b表达明显降低(p < 0.001)，而且和对照组相比17P-，t(4;14)异位，和死亡患者中miRNA-33b表达明显降低(p = 0.032, 0.018, 0.034, 0.005)。 .2. miR-33b 低表达患者有着更低的PFS (p = 0.016) 和 OS (p = 0.033) ，但并非独立预后因素，并且对硼替佐米为基础的治疗耐药。.3..在MM患者和细胞系中制作miRNA-33b的FISH探针检测，均提示为正常杂交信号。.4..使用甲基化特异性PCR定量检测MM患者、正常人CD138+和MM细胞系，结果显示MM细胞系和患者miRNA-33b启动子区域甲基化较正常人高。.5..对六株MM细胞系（8226，H929，U266，MM1S，ARP1和OCI）行P53 FISH检测显示均存在P53高比例杂合型缺失。.6..转染miRNA-33b至U266细胞，转染后72h， MDM2蛋白表达显著下降，P53及其下游蛋白P21表达明显增加。.7.本研究证明了miRNA-33b在MM患者的低表达，并且和高危遗传学异常相关，miRNA-33b低表达的患者可能对硼替佐米为基础的治疗耐药，可作为有价值的预后分子标记。miRNA-33b降低了抑癌基因P53及其下游蛋白P21表达，从而促进MM细胞生长。