Nishioka Chie, Ikezoe Takayuki, Yang Jing, Yokoyama Akihito
Department of hematology and Respiratory Medicine immunology, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan.
Department of immunology, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan.
PLoS One. 2015 May 8;10(5):e0125017. doi: 10.1371/journal.pone.0125017. eCollection 2015.
We recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34(+)/CD38- acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34(+)/CD38(-) AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression.
We employed microarray analysis comparing the gene expression profiles between CD34(+)/CD38(-) AML cells transduced with CD82 shRNA and CD34(+)/CD38(-) AML cells transduced with control shRNA. Real-time RT-PCR and western blot analysis were performed to examine the effect of CD82 knockdown on the expression of the polycomb group member, enhancer of zeste homolog 2 (EZH2), in leukemia cells. A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of EZH2 bound to the promoter regions of tumor suppressor genes in leukemia cells. We also utilized methylation-specific PCR to examine whether CD82 expression influences the methylation status of the tumor suppressor gene promoter regions in leukemia cells.
Microarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34(+)/CD38(-) AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.
我们最近发现，四跨膜蛋白家族成员CD82在化疗耐药的CD34(+)/CD38-急性髓系白血病（AML）细胞中异常表达，它对基质金属蛋白酶9起负性调节作用，并在使CD34(+)/CD38(-) AML细胞黏附于骨髓微环境中发挥重要作用。本研究探讨了有助于AML进展的CD82的新功能。
我们采用微阵列分析比较用CD82短发夹RNA（shRNA）转导的CD34(+)/CD38(-) AML细胞和用对照shRNA转导的CD34(+)/CD38(-) AML细胞之间的基因表达谱。进行实时逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析，以检测CD82敲低对白血病细胞中多梳蛋白家族成员、zeste同源物2增强子（EZH2）表达的影响。进行染色质免疫沉淀试验，以检测CD82表达对白血病细胞中与肿瘤抑制基因启动子区域结合的EZH2量的影响。我们还利用甲基化特异性PCR检测CD82表达是否影响白血病细胞中肿瘤抑制基因启动子区域的甲基化状态。
微阵列分析显示，在CD34(+)/CD38(-) AML细胞中，shRNA介导的CD82缺失后EZH2水平降低。此外，白血病细胞中抗体介导的CD82阻断通过激活p38丝裂原活化蛋白激酶（MAPK）信号通路降低EZH2表达，减少与肿瘤抑制基因启动子区域结合的EZH2量，并抑制这些启动子区域中的组蛋白H3赖氨酸27三甲基化，导致肿瘤抑制因子在mRNA和蛋白质水平均上调。
