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

H3K27me3 aggregate promoter of MEG3 lncRNA and induce apoptosis escape of RPMI8226 cell through MDM2/p53 pathway

目的从组蛋白甲基化与长链非编码RNA(lncRNA)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;Annexin V-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RPMI8226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RPMI8226细胞中Zeste基因增强子同源物2(EZH2)、H3K27me3、MDM2及p53蛋白表达;Ch IP 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;RPMI8226经MEG3lncRNA小干扰RNA(MEG3lncRNA-siRNA)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 lncRNA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。

Objective To investigate the interaction of lncRNA and aberrant histone modification in multiple myeloma 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）,MEG3 lncRNA,murine double minute 2（MDM2） and p53.The bonding site of promoter of MEG3 by H3K27me3 was confirmed by Ch IP-Real-time PCR,shRNA interference was used to downregulate H3K27me3 through inhibiting the expression of EZH2.Results The expression of H3K27me3 could be significantly inhibited by interfering EZH2,which indicating downregulation of H3K27me3 could induce apoptosis of RPMI8226 cells.H3K27me3 protein could bind to MEG3 lncRNA promoter directly.H3K27me3 inhibition could increase the expression of MEG3 lncRNA.Furthermore,MEG3 lncRNA could downregulated by MEG3 lncRNA-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.