the effects of promoter methylation on downregulation of dazap2 in multiple myeloma cell lines启动子甲基化的影响的差别在对这些dazap2在多发性骨髓瘤细胞系.pdfVIP

The Effects of Promoter Methylation on Downregulation of DAZAP2 in Multiple Myeloma Cell Lines . . Sai-Qun Luo , Jing-Ping Hu , Qiang Qu, Jiang Li, Wei Ren, Jia-Ming Zhang, Yan Zhong, Wei-Xin Hu* Molecular Biology Research Center, Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China Abstract Our previous studies had shown that DAZAP2 was profoundly downregulated in bone marrow mononuclear cells from multiple myeloma patients. In this report, we analyzed epigenetic changes in multiple myeloma cell lines to understand the molecular mechanisms underlying the downregulation of DAZAP2. Four multiple myeloma cell lines, KM3, MM.1S, OPM-2 and ARH-77, were studied. The results of methylation specific PCR (MSP) showed that the promoter of DAZAP2 was methylated for KM3, MM.1S, OPM-2 and unmethylated for ARH-77. The DAZAP2 promoter region was amplified to obtain a series of different length sequences. All of the amplified sequences were inserted to luciferase reporter vector. The constructs were transfected into COS-7 cells and the luciferase activities were measured to search for the core region of DAZAP2 promoter. Two CpG islands were found in DAZAP2 promoter region. The results of luciferase assay showed that CpG island 1 displayed weak transcriptional activity, whereas CpG island 2 exhibited strong transcriptional activity (273 folds) compared to the control. The sequence that covered both CpG islands 1 and 2 showed higher activity (1,734 folds) compared to the control, suggesting that the two islands had synergistic effect on regulating DAZAP2 expression. We also found that M. Sss I methylase could inhibit the luciferase activity, whereas demethylation using 5-aza-29-deoxycytidine treatment rescued the e