转录因子E4BP4在多发性骨髓瘤中的作用及分子机制研究

Multiple myeloma(MM) is a high incidence of malignant tumor in blood system, transcription factor is important role on the occurrence and development of MM. In our preliminary study, we found E4BP4 highly expressed in clinical MM samples and MM cell line U266、IM9 and RPMI8226. Glucocorticoid treatment could down-regulate the expression of E4BP4 and modulate apoptosis in U266 and RPMI8226, which indicates that E4BP4 plays a critical role in MM. In this project, we intended to enroll more clinical MM patients to investigate the association between E4BP4 and various of clinical related outcomes of MM and make clear of the identity of E4BP4 as a tumor related protein both in vitro and vivo. We will treat nude mice with E4BP4 recombinant protein or lentivirus stable cell lines to explore the role of E4BP4 in tumor progression. Furthermore, We ought to develop a tandem affinity purification (TAP) methods coupled with mass spectrometry and co-immunoprecipitation to detect the possible interaction proteins of endogenous E4BP4. We also wish to investigate the detail molecular mechanisms for E4BP4 in MM progression, analysis its regulation, target substrate and involving signal pathway both in vivo and in vitro. This research will provide important clue to discover new molecular target drug for MM therapy.

多发性骨髓瘤（MM）是血液系统高发的恶性肿瘤，其发生发展与转录因子的异常调节密切相关。本课题组在前期研究中发现转录因子E4BP4在MM患者骨髓细胞和MM细胞系U266、IM9、KM3、RPMI8226中表达增高，加入糖皮质激素（GC）干预后，U266、RPMI8226中E4BP4表达下降，并且GC可以诱导U266、RPMI8226凋亡，上述结果提示我们E4BP4可能参与MM的发生，并且GC对MM的治疗可能通过E4BP4发挥重要作用。本项目拟扩大临床MM样本明确E4BP4与MM病人各项临床指标的关系；通过穿膜侵袭和裸鼠成瘤等实验鉴定E4BP4肿瘤相关蛋白的身份；拟建立串联标签标记内源性E4BP4并结合免疫沉淀和质谱分析鉴定其相互作用蛋白，从细胞、动物和临床病人深入研究E4BP4在MM发生中的上游调控机制、下游功能底物及主要信号通路，揭示其在MM中的作用及分子机理，为MM的诊治提供实验依据。

本课题按照预定的研究计划，发现转录因子E4BP4 与正常人外周血单个核细胞比较，mRNA在多发性骨髓瘤（MM）细胞株和病人中高表达，E4BP4蛋白在MM细胞株和病人中高表达，并且发现III期MM病人的E4BP4 mRNA高于MM I或II期患者的水平，表明E4BP4参与了MM病人进展。但具体的作用机制如何？本课题通过成功构建过表、沉默E4BP4的MM细胞株，发现E4BP4促进MM细胞克隆形成、促进MM细胞体外增殖，同时抑制凋亡， 为了进一步研究其机制，我们通过数据库检索分析发现E4PB4与HDAC3呈显著正相关，而在MM细胞株中敲低E4BP4发现HDAC3表达减少，而过表E4BP4后HDAC3表达明显增加，免疫荧光发现二者在胞浆胞核均有表达，且胞核表达大于胞浆，通过双荧光素梅基因报告实验和CoIP发现二者存在相互作用，结果提示E4PB4通过调控HDAC3影响MM的发生发展，实验已经完成并进入投稿阶段，待正式发表后我们将导入成果目录中审核。对研究计划中收集的MM临床病例，我们将继续追踪，观察MM与E4PB4的预后关系，为E4PB4的临床转化收集证据，并将对这部分数据进行分析总结，为E4PB4的进一步研究和转化奠定基础。