多发性骨髓瘤细胞中DAZAP2基因启动子甲基化对p38 MAPK/NF-κB信号转导途径影响的机制研究

We found that DAZAP2 gene in multiple myeloma (MM) cells was significantly down-regulated in our previous work. In order to clarify the mechanism of DAZAP2 down-regulation, the effects of DAZAP2 DNA epigenetic changes on gene expression were analyzed. We found that the hyper-methylation in DAZAP2 promoter was strongly correlated to down-regulation of gene expression. The results revealed that the methylation of promoter in MM cells could play an important role in DAZAP2 down-regulation. There were transcriptional binding sites of c-Rel (a member of NF-κB family) and CREB (both were downstream effectors of p38 MAPK) in the CpG island 2 of DAZAP2 promoter in analysis of bioinformatics, and the bindings between transcription factors and binding sites were affected by the methylation in our preliminary experiments. The mechanism of DAZAP2 down-regulation in MM pathogenesis will be further illustrated in this project. We will make clear that the bindings of transcription factors CREB and c-Rel can be inhibited by methylation of CpG islands in DAZAP2 promoter, finally lead to the DAZAP2 down-regulation. The effects of p38 MAPK signaling and NF-κB signaling on DAZAP2 gene expression will be elucidated in MM cells. It is possible to provide a new insight for methods of MM diagnosis and treatment by this project.

前期的研究发现，MM细胞中DAZAP2基因表达显著下调。为阐明DAZAP2基因下调机制，分析了DAZAP2基因表观遗传学变化对基因表达的影响。发现DAZAP2启动子的高甲基化与其表达下调显著相关，证明MM中DAZAP2基因启动子的甲基化在其表达下调中起到十分重要的作用。生物信息学分析发现，DAZAP2启动子CpG岛2有一个c-Rel（NF-κB家族成员）和一个CREB转录因子结合位点，二者都是p38 MAPK下游效应分子，甲基化影响c-Rel和CREB与其结合。通过本研究，进一步获得DAZAP2表达缺失在MM发病中的作用机制。明确MM细胞中DAZAP2启动子甲基化后抑制了转录因子CREB和c-Rel与CpG岛的结合，导致DAZAP2基因表达下调。阐明MM细胞中p38 MAPK信号通路和NF-κB信号通路对DAZAP2基因表达调节机制，为建立MM新的诊断方法和治疗措施，提供新的思路。

前期的研究发现，DAZAP2是一个多发性骨髓瘤（MM）负性相关基因，在MM患者和MM细胞株中表达显著下调。DAZAP2基因启动子区高度甲基化在DAZAP2基因表达下调中发挥重要作用。MM细胞中，DAZAP2基因由于启动子区高度甲基化而表达下调。生物信息学分析结果显示DAZAP2基因启动子区的CpG岛2存在转录因子CREB结合位点，且该位点受到了甲基化的影响。CREB是p38/MAPK信号通路的下游效应分子。为了阐明DAZAP2基因启动子区甲基化对p38/MAPK信号通路调控DAZAP2表达的影响，我们采用EMSA、ChIP结合Q-PCR、RNA干扰等方法检测CREB对DAZAP2基因的表达调控，并分析p38/MAPK信号通路在此调控过程中的作用。结果显示，p38/MAPK信号通路参与了细胞内DAZAP2基因的表达调控，这种调控作用是通过其下游效应分子CREB的磷酸化而实现的。而启动子区的高度甲基化使得转录因子CREB无法与DAZAP2基因启动子区域结合，从而阻断了p38/MAPK信号通路对DAZAP2基因表达的调控，导致了在MM细胞中DAZAP2基因低表达。通过本研究，进一步获得DAZAP2表达缺失在MM发病中的作用机制。阐明了MM细胞中p38 MAPK信号通路对DAZAP2基因表达调节机制，为建立MM新的诊断方法和治疗措施，提供新的思路。.前期研究也发现，C1orf35是一个与MM恶性相关的基因。在MM细胞中，C1orf35与癌基因c-MYC的表达呈正相关，且C1orf35可调控MM细胞中c-MYC的表达。为了阐明C1orf35在MM中激活c-MYC表达的分子机制，我们采用EMSA、ChIP结合Q-PCR、RNA干扰、过表达、RIP结合RT-qPCR、LC-MS等方法，发现C1orf35可在转录水平和转录后水平参与调控MM细胞内c-MYC的表达，并影响细胞的代谢。