H3K27me3聚集MEG3 lncRNA启动子通过MDM2/p53途径诱导多发性骨髓瘤RPMI8226细胞凋亡逃逸

摘要

Objective To investigate the interaction of lncRNA and aberrant histone modification in multiple mye-loma cells escaping from apoptosis. Methods Cell proliferation activity was tested by MTT; Annexin V-FITC/PI dual staining was used to test apoptotic cells, RT-PCR was used to test the mRNA expression of enhancer of Zeste homolog 2(EZH2), MEG3lncRNA, murine double minute 2 (MDM2) and p53. The bonding site of promoter of MEG3 by H3K27me3 was confirmed by ChIP-Real-time PCR, shRNA interference was used to downregulate H3K27me3 through inhibiting the expression of EZH2. Results The expression of H3K27me3 could be signifi-cantly inhibited by interfering EZH2, which indicating downregulation of H3K27me3 could induce apoptosis of RP-MI8226 cells. H3K27me3 protein could bind to MEG3lncRNA promoter directly. H3K27me3 inhibition could in-crease the expression of MEG3lncRNA. Furthermore, MEG3lncRNA could downregulated by MEG3lncRNA-siRNA in RPMI8226 cells, meanwhile, both protein and mRNA of MDM2 increased in cells. Ubiquitination of p53 was founded upregulation and p53 degradation when RPMI8226 cells incubated with MDM2 inhibitor. Conclusion H3K27me3 highly expresses in RPMI8226 multiple myeloma cells, which cause loss of MEG3 lncRNA. The loss expression of MEG3 lncRNA could paralyze the inhibition of MDM2, promote the degradation or inaction of p53 protein. This could be one of the mechanisms of multiple myeloma cells escape from apoptosis.%目的 从组蛋白甲基化与长链非编码 RNA ( ln-cRNA)相互作用角度探讨多发性骨髓瘤( MM)细胞凋亡逃逸的具体机制.方法 MTT法检测细胞增殖活性;Annex-inV-FITC/PI双标流式细胞术检测下调组蛋白H3第27位赖氨酸上三甲基化H3K27me3 的表达是否可以诱导多发性骨髓瘤细胞株—RPMI8226 细胞凋亡; RT-PCR 法检测 RP-MI8226细胞中人母系表达基因( MEG3) lncRNA及鼠双微体基因 2 ( MDM2 )的 mRNA 表达; Western blot 检测 RP-MI8226 细胞中 Zeste 基因增强子同源物 2 ( EZH2 )、H3K27me3、MDM2 及 p53 蛋白表达; ChIP Real -time PCR检测RPMI8226细胞中 H3K27me3 的结合位点.结果 在RPMI8226 细胞中,下调 H3K27me3 可诱导细胞凋亡;H3K27me3可直接结合于 MEG3lncRNA 启动子; RPMI8226细胞中抑制了 H3K27me3 可上调 MEG3lncRNA 表达; RP-MI8226经MEG3lncRNA 小干扰 RNA ( MEG3lncRNA -siR-NA)干预下调MEG3lncRNA时,细胞内MDM2 mRNA水平明显升高,并诱导 MDM2 蛋白表达.给予 MDM2 拮抗剂后, Ub-p53 表达降低, p53 降解被抑制.结论 MM 中H3K27me3结合MEG3 lncRNA启动子,MEG3lncRNA启动子H3K27me3高表达导致MEG3 lncRNA表达缺失. MEG3 ln-cRNA表达缺失解除MDM2的抑制, MDM2与p53直接结合介导p53泛素化,促使p53降解,细胞凋亡逃逸.