Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma
Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma
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DOI：
10.1158/1078-0432.ccr-08-3336
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发表时间：
2009-07-01
影响因子：
11.5
通讯作者：
Shinomura, Yasuhisa
中科院分区：
文献类型：
--
作者：
Nojima, Masanori;Maruyama, Reo;Shinomura, Yasuhisa
目的：DNA甲基化等表观遗传学改变在多发性骨髓瘤的发生发展中起关键作用。我们在本研究中的目的是利用基因组筛选来确定多发性骨髓瘤中表观遗传失活的靶基因，并评估它们在地塞米松耐药中的作用。实验设计：采用微阵列筛选、逆转录-聚合酶链式反应和实时定量聚合酶链式反应检测基因表达。采用亚硫酸氢盐聚合酶链式反应、亚硫酸氢盐测序法和亚硫酸氢盐焦磷酸测序法对14例多发性骨髓瘤细胞系、87例多发性骨髓瘤标本和12例对照骨髓标本进行DNA甲基化检测。WST-8法检测经5-氮-2‘-脱氧胞苷和/或地塞米松处理后的细胞存活率。结果：应用基因芯片技术筛选5-氮-2’-脱氧胞苷上调的基因。RPMI8226细胞中有128个基因表达上调，而KMS12PE细胞中有83个基因表达上调。22个基因的5‘端有CpG岛甲基化，包括RASD1。RASD1基因甲基化与其失活有关，失活与地塞米松耐药有关。用5-氮-2‘-脱氧胞苷处理多发性骨髓瘤细胞可恢复对地塞米松的敏感性。在部分原发多发性骨髓瘤标本中也检测到RASD1的甲基化，在反复抗肿瘤治疗后甲基化水平增加。基因信号分析显示，5-氮-2‘-脱氧胞苷和地塞米松联合治疗可协同诱导多种基因。结论：包括RASD1在内的基因表观遗传失活在多发性骨髓瘤地塞米松耐药的形成中起关键作用。此外，它们还显示了去甲基化治疗在晚期多发性骨髓瘤病例中的有效性。
Purpose: Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone.Experimental Design: Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples. WST-8 assays were used to assess cell viability after treatment with 5-aza-2'-deoxycytidine and/or dexamethasone.Results: Microarray analysis was done to screen for genes up-regulated by 5-aza-2'-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5' regions, including RASD1, was confirmed. Methylation of RASD1 was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2'-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2'-deoxycytidine plus dexamethasone.Conclusion: Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.