MiRNA-15a/-16靶向调控PHF19基因对多发性骨髓瘤细胞增殖和耐药的影响及机制研究

Although major progress has been made in the treatment of MM with an increase in median survival from 2.5 to 10 years over the last two decades, the large majority of the patients still relapse and ultimately die of their disease. This is due to the persistence of a drug-resistant subpopulation of MM cells even after intensive therapy with tandem autologous transplants and prolonged maintenance with the newer drugs. To achieve a cure for MM, a better understanding of the genetic makeup of these drug-resistant MM cells is required so that these cells can be specifically targeted. Our previous study of sequential analysis of gene expression profiles (GEP) of tumor cells in MM patients showed that PHF19 expression was significantly up-regulated in resistant tumor cell clones of pre-1st ASCT and relapse MM patients. Kaplan-Meier analyses showed that MM patients with higher expression of PHF19 had a significantly inferior PFS and OS. Our preliminary results also demonstrated that miRNA-15a/-16 had lower expression in MM cells. Dual luciferase reporter assay indicated that PHF19 was the target gene of miRNA-15a/-16. Up-regulation of miRNA-15a/-16 suppressed MM cells proliferation and promoted drug induced apoptosis. It is our hypothesis that PHF19 as a target gene of miRNA-15a/-16 promotes MM cells proliferation and drug-resistance. Therefore, this project will investigate the miRNA-15a/-16 mediates PHF19 functions of cell proliferation and drug resistance in MM. Combined with ChIP-seq and microarray high-throughput assay, Co-IP experiments, as well as 5TGM1 mouse MM model, the PHF19 key signaling pathways and the role of regulatory molecules and epigenetic modification of information will be clarified. The results will provide theoretical and experimental foundation for the search for new therapeutic targets in MM.

MM依然是一种不可治愈的恶性肿瘤，耐药是导致复发的重要原因。探明MM复发、耐药的分子机制，对提高MM疗效具有重要意义。课题组前期对MM患者细胞序贯基因表达谱分析发现PHF19在治疗后及复发时肿瘤耐药克隆中表达明显增高并具有临床预后意义。并且证实miRNA-15a/-16靶向调控PHF19表达，上调miRNA-15a/-16表达可抑制MM细胞增殖、提高药物敏感性。据此我们提出MM细胞低表达miRNA-15a/-16导致其靶基因PHF19表达增加，促进MM细胞增殖与耐药，导致患者生存期缩短，预后不良的科学假设。本研究拟从基因、蛋白水平，体外及体内实验等多层次探讨miRNA-15a/-16靶向调控PHF19以及PHF19促进MM细胞增殖和耐药的分子机制。并结合ChIP-seq等高通量检测方法，探明PHF19作用的关键信号通路及调控分子和表观遗传学修饰信息，为寻找新的治疗靶点提供理论及实验基础。

MM依然是一种不可治愈的恶性肿瘤，耐药是导致复发的重要原因。探明MM复发、耐药的分子机制，对提高MM疗效具有重要意义。课题组前期对临床MM患者细胞序贯基因表达谱分析发现PHF19在治疗后及复发时肿瘤耐药克隆中表达明显增高并具有临床预后意义。生物信息学分析提示miRNA-15a/-16靶向调控PHF19表达，上调miRNA-15a/-16表达可抑制MM细胞增殖、提高药物敏感性。据此我们提出MM细胞低表达miRNA-15a/-16导致其靶基因PHF19表达增加，促进MM细胞增殖与耐药，导致患者生存期缩短，预后不良的科学假设。本研究首先应用双荧光素酶报告基因系统证实miR-15a与PHF19基因 3’UTR区直接结合，降解PHF19 mRNA，从而抑制其mRNA合成及蛋白翻译，MM细胞低表达miR-15a是导致其PHF19表达升高的重要原因。为了阐明PHF19在介导MM细胞增殖存活、耐药中的作用机制，应用慢病毒感染系统制备PHF19过表达的MM细胞系，体外增殖曲线、软琼脂克隆形成实验及流式细胞术检测显示PHF19过表达后MM细胞增殖速度加快、克隆形成能力增强，对万坷、多柔比星、依托泊甙等化疗药物敏感性降低。利用NOD/SCID小鼠模型，也证实PHF19过表达MM细胞体内增殖能力明显加强，对万坷等药物治疗耐药，PHF19过表达组小鼠生存时间明显缩短。采用条件性诱导敲除PHF19表达后，细胞增殖明显受抑、对化疗药物敏感性增加，且小鼠生存期得到延长。进一步采用TAP/MASS、CoIP、RNAseq、Western Blot等技术研究发现PHF19可与EZH2/AKT等蛋白结合，促进磷酸化EZH2水平，使其H3K27甲基化酶活性降低，因此PHF19过表达细胞中H3K27me3水平降低，促进HIF1a、IGF1等蛋白表达上升，从而促进并介导MM细胞增殖加快及耐药的产生。抑制PHF19表达可逆转MM细胞增殖加快及耐药的产生。本研究从基因、蛋白水平，体外及体内实验等多层次阐明miRNA-15a靶向调控PHF19以及PHF19促进MM细胞增殖和耐药的分子机制。为开发以PHF19为治疗靶点的新的治疗策略提供理论及实验基础。