DNA甲基化介导miR-203/CREB1信号调控对多发性骨髓瘤细胞增殖及凋亡的影响  

Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells

作　　者：徐成波[1] 廖斌[1] 付海英[2] 齐彦[1] 沈建箴[2] XU Cheng-Bo;LIAO Bin;FU Hai-Ying;QI Yan;SHEN Jian-Zhen(Department of Hematology,The Affiliated People’s Hospital to Fujian University of Traditional Chinese Medicine,Fuzhou 350004,Fujian Province,China;Department of Hematology,Fujian Medical University Union Hospital,Fujian Institute of Hematology,Fuzhou 350001,Fujian Province,China)

机构地区：[1]福建省人民医院福建中医药大学附属人民医院血液科,福建福州350004 [2]福建医科大学附属协和医院血液科福建省血液病研究所,福建福州350001

出　　处：《中国实验血液学杂志》2022年第3期790-796,共7页Journal of Experimental Hematology

基　　金：福建省教育厅中青年教师教育科研项目(JAT202215);福建省自然科学基金项目(2021J01897);福建省卫生健康中青年骨干人才培养项目(2021GGA059)。

摘　　要：目的:探讨DNA甲基化介导miR-203/CREB1信号途径的调控对多发性骨髓瘤细胞增殖、侵袭和凋亡等生物学行为的影响。方法:采用重亚硫酸氢盐测序法定量检测多发性骨髓瘤RPMI 8226细胞株中miR-203的甲基化水平;实时荧光定量PCR检测miR-203的表达水平;应用5-杂氮-2’-脱氧胞苷(5-Aza-CdR)干预RPMI 8226细胞建立去甲基化模型;转染miR-203模拟物(mimic)构建miR-203过表达细胞;采用CCK-8法、Transwell、流式细胞术分别检测RPMI 8226细胞增殖、侵袭和凋亡;采用双荧光素酶报告实验验证miR-203与CREB1的靶向作用关系;Western blot检测CREB1的蛋白表达水平。结果:RPMI 8226细胞miR-203启动子区存在高甲基化且低表达,去甲基化可以诱导表达;去甲基化作用可以抑制RPMI 8226细胞增殖及细胞侵袭能力,促进细胞凋亡,与空白对照组比较差异有统计学意义(P<0.05)。miR-203过表达组的细胞增殖抑制作用、细胞侵袭能力、凋亡率与去甲基化作用组相当。双荧光素酶报告实验证实,CREB1是miR-203的直接作用靶点;去甲基化作用通过上调miR-203抑制CREB1蛋白的表达水平,与对照组比较差异有统计学意义(P<0.05)。结论:miR-203甲基化沉默导致CREB1表达上调是维持多发性骨髓瘤RPMI 8226细胞恶性生物学行为的重要原因。Objective: To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation o n the proliferation, invasion and apoptosis of multiple myeloma(MM) cells. Methods: The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction(BSP). The mRNA e xpression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine(5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between m iR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of C REB1 protein. Results: Hypermethylation of miR-203 promoter region and lowexpression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The p roliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical s ignificance as compared with blank control group(both P<0.05),while the apoptosis rate was promoted(P<0.05). The p roliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibitedb y demethylation and showed statistical significance as compared with control group(P<0.05). Conclusion: MiR-203 t argeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

关 键 词：甲基化 miR-203 环腺苷酸反应元件结合蛋白1 多发性骨髓瘤 凋亡 

分 类 号：R733.3[医药卫生—肿瘤]