Objective: To investigate the expression, mechanism and methylation level of miR-28-5p in multiple myeloma (MM), so as to provide the expirement basis for searching new targeted therapy.
Methods: RT-PCR was used to detect the expression levels of miR-28-5p and potential target CCND1 in CD138+ cells of the patients with MM and bone marrow mononuclear cells of patients with iron defficiency anemia(IDA) as control, Methylation-specific PCR(MSP) was used to detect methylation levels of CpG island in LPP/miR-28-5p promoter region and the correlation with other clinical indicators was analyzed. The 5-aza-2'-deoxycytidine (5-Aza-dC，DAC) was used to treat MM cell line U266; after drug treatment，MSP was used to analyze the methylation status of the CpG islands in LPP/miR-28-5p promoter; the qPCR was used to detect the expression levels of miR-28-5p，and the regulatory mechanism of miR-28-5p expression was explored furtherly.
Results: The methylation level of CpG island in LPP/miR-28-5p promoter region of MM patients was significantly higher than that of IDA patients. The relative expression level of miR-28-5p in MM patients was significantly lower than that of IDA patients. The relative expression level of miR-28-5p in newly diagnosed MM patients was higher than that in relapsed/progressive patients. The miR-28-5p target CCND1 was expressed at high levels in MM patients with LPP / miR-28-5p methylation, the expression level of miR-28-5p in MM patients correlated with β2-MG concentration. 5-aza-dc could significantly inhibit the growth of U266 cell line, arrest the cell cycle in G1 phase, inhibit the biosynthesis of cellular RNA and protein and promote cell apoptosis. At the same time, up-regulation of miR-28-5p expression was found.
Conclusion: The expression of miR-28-5p in MM patients is regulated by methylation of CpG islands in the promoter region of the genome.miR-28-5p may act as a tumor suppressor gene, and its low expression may be involved in the occurrence and development of MM, suggesting that miR-28-5p may become a new target for the treatment of MM.
题目: microRNA-28-5p在多发性骨髓瘤的表达分析与表观遗传学研究.
目的: 探讨miR-28-5p在多发性骨髓瘤（MM）中的表达、作用机制及其甲基化水平在MM诊断中的应用，为寻求新的靶向治疗方法提供依据.
方法: 采用RT-PCR检测MM患者骨髓单个核CD138+细胞和相关疾病对照组（缺铁性贫血患者）骨髓单个核细胞的miR-28-5p及miR-28-5p潜在靶标CCND1的表达水平；甲基化特异性PCR(MSP)检测LPP/miR-28-5p启动子区 CpG岛甲基化水平，并分析与其他临床指标的相关性。用甲基转移酶抑制剂 5-aza-dc对多发性骨髓瘤细胞株U266进行处理，用MSP对药物处理后细胞内LPP/miR-28-5p启动子区 CpG 岛的甲基化状态进行分析；应用qPCR检测药物处理后细胞内miR-28-5p的表达水平，并探讨miR-28-5p表达的调控机制.
结果: MM患者LPP/miR-28-5p启动子区 CpG 岛甲基化水平明显高于IDA对照者，MM患者miR-28-5p的相对表达量较IDA对照者明显降低，初诊MM患者miR-28-5p 的相对表达水平高于复发/进展期患者,已知的miR-28-5p靶标CCND1在具有LPP / miR-28-5p甲基化的MM患者中以高水平表达，MM患者miR-28-5p的表达水平与β2-MG水平呈相关性；5-aza-dC 能够显著地抑制U266细胞株的生长，使细胞周期停滞于 G1期，抑制细胞ＲNA和蛋白质的生物合成，促进细胞凋亡，同时能上调miR-28-5p的表达.
结论: 在MM患者中，miR-28-5p的表达受基因组启动子区CpG岛甲基化修饰的调控，miR-28-5p可作为抑癌基因，其低表达可能参与MM的发生、发展，提示miR-28-5p可能成为治疗MM的新靶向.