PHF19 Promotes Cell Growth and Confers Drug Resistance through EZH2 Phosphorylation in Multiple Myeloma

To determine the effect of overexpression PHF19 in ARP1 myeloma cells Drug resistance is a major reason for the recurrence of multiple myeloma (MM). In addition to genetic mutations, epigenetic abnormalities are involved in the whole process of MM relapse and drug resistance. In this study, we investigate the roles of polycomb like protein 19 (PHF19), a critical gene involved in the epigenetic regulation of gene expression, in the development of MM. The gene expression profiling (GEP) data shows that PHF19 is significantly up-regulated in MM cells and associated with shortened progression-free survival and overall survival in MM patients. PHF19 promotes MM cell growth both in vitro and in a subcutaneous xenograft mouse model. Up-regulated PHF19 is also associated with the development of resistance to multiple drugs, including bortezomib, in MM cells. Moreover, miR-15a can directly target PHF19 by binding to its 3'-UTR. Since miR-15a is down-regulated in MM cells, PHF19 is considered to be up-regulated due to loss of the suppression by miR-15a. The mechanistic investigations indicate that PHF19 can activate AKT which in turn leads to an increase in the phosphorylation of EZH2, a core component of PRC2 that mediates the histone H3K27 methylation. Subsequently suppresses histone H3K27 methylation and causes up-regulated expression of several anti-apoptotic genes, including BCL-xL, MCL-1 and HIF-1alpha. Moreover, PDK1 activation is significantly increased in PHF19 overexpressing MM cells. ARP1 cells were transfected by lenti-virus plasmids with PHF19 ORF, then PHF19 overexpressed cells were sorted. And RNA was extracted.

为探究PHF19在ARP1骨髓瘤细胞中的过表达效应，本研究首先明确：耐药性是多发性骨髓瘤（multiple myeloma, MM）复发的核心诱因。除遗传突变外，表观遗传异常全程参与多发性骨髓瘤的复发与耐药进程。多梳样蛋白19（Polycomb Like Protein 19, PHF19）是参与基因表达表观调控的关键基因，本研究旨在解析其在多发性骨髓瘤发生发展中的作用。基因表达谱（gene expression profiling, GEP）数据显示，PHF19在多发性骨髓瘤细胞中显著上调，且与患者的无进展生存期与总生存期缩短显著相关。PHF19可在体外及皮下移植瘤小鼠模型中促进多发性骨髓瘤细胞增殖。在多发性骨髓瘤细胞中，PHF19上调还与包括硼替佐米（bortezomib）在内的多种药物耐药性的产生密切相关。此外，miR-15a可通过结合PHF19的3'非翻译区（3'-UTR）直接靶向调控该基因。由于miR-15a在多发性骨髓瘤细胞中表达下调，因此PHF19的上调被认为是由于丧失了miR-15a的负向调控。机制研究显示，PHF19可激活AKT信号通路，进而促使PRC2（Polycomb Repressive Complex 2, PRC2）的核心组分EZH2（Enhancer of Zeste Homolog 2, EZH2）的磷酸化水平升高——PRC2是介导组蛋白H3K27甲基化的关键复合体。该通路后续可抑制组蛋白H3K27甲基化，进而上调BCL-xL、MCL-1及HIF-1α等多种抗凋亡基因的表达。此外，在PHF19过表达的多发性骨髓瘤细胞中，PDK1（Pyruvate Dehydrogenase Kinase 1, PDK1）的激活水平显著升高。本研究通过携带PHF19开放阅读框（open reading frame, ORF）的慢病毒质粒转染ARP1细胞，随后筛选获得PHF19过表达细胞株，并提取其总RNA。