双链RNA通过激活PKR/eIF2α通路抑制恶性胶质瘤生长和迁移  

Double-stranded RNA inhibits the growth and migration of malignant gliomas through the activation of the PKR/eIF2αpathway

作　　者：燕群 卞莎莎 束敏峰 YAN Qun;BIAN Sha-sha;SHU Min-feng(Department of Pharmacology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学基础医学院药理学系,上海200032

出　　处：《复旦学报（医学版）》2024年第3期295-305,共11页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81872467,82073880)。

摘　　要：目的研究放疗模拟剂博来霉素(Zeocin)诱导的双链RNA(double-stranded RNA,dsRNA)对PKR(double-stranded RNA-dependent protein kinase)通路激活和RNA m6A相关酶的影响,从而探索其抑制恶性胶质瘤生长和迁移的分子机制。方法通过划痕实验、平板克隆形成实验、CCK8实验检测Zeocin处理后的恶性胶质瘤细胞生长和迁移情况;通过Western blot和RT-qPCR实验检测甲基修饰酶的mRNA表达水平;使用J2抗体检测Zeocin处理胶质瘤细胞后细胞内dsRNA的产生水平;利用Western blot实验检测相关甲基修饰酶的表达水平、双链RNA依赖的蛋白激酶R(double-stranded RNA-dependent protein kinase,PKR)及真核生物起始因子2α(eukaryotic initiating factor 2α,eIF2α)的磷酸化水平。结果放疗模拟剂Zeocin显著抑制恶性胶质瘤细胞的生长和迁移;Zeocin处理恶性胶质瘤细胞可产生内源的双链RNA增多;随着Zeocin浓度的增加,磷酸化的PKR与eIF2α水平均显著上升;Zeocin可以剂量依赖性下调胶质瘤细胞内总m6A水平;Zeocin可以选择性下调甲基化酶METTL14的蛋白水平;Zeocin对m6A相关酶的mRNA水平无影响。结论Zeocin可能通过下调恶性胶质瘤细胞中RNA的m6A水平,产生较多的dsRNA,进而激活PKR/eIF2α免疫调节通路,最终导致肿瘤生长抑制。Objective To investigate the impact of radiotherapy simulant Zeocin induced double-stranded RNA(dsRNA)on double-stranded RNA-dependent protein kinase(PKR)activation and explore its molecular mechanism in inhibiting the growth and migration of malignant gliomas.Methods We employed scratch assays,colony formation assays,and CCK8 assays to assess the impact of Zeocin on glioma growth and migration inhibition.Western blot and RT-qPCR were employed to measure the expression levels of methyl-modifying enzymes in glioma cells.The J2 antibody was used to detect endogenous dsRNA in malignant glioma cells treated with Zeocin.Western blot was used to assess the phosphorylation levels of PKR and eukaryotic initiating factor 2α(eIF2α).Results Zeocin significantly inhibited growth and migration of glioma cells;Zeocin treatment induced the production of endogenous dsRNA in malignant glioma cells;as the concentration of Zeocin increased,phosphorylated PKR and eIF2αprotein level also significantly increased;Zeocin downregulated the total m6A level of glioma cells in a dose-dependent manner;Zeocin selectively downregulated the protein level of methylase METTL14;Zeocin had no effect on mRNA levels of m6A modifying enzymes.Conclusion Zeocin probably triggered more dsRNA by downregulating the m6A level of RNA in malignant glioma cells,which in turn activated the PKR/eIF2αpathway,ultimately leading to tumor growth inhibition.

关 键 词：胶质瘤 m6A甲基化 RNA修饰 双链RNA PKR 

分 类 号：R739.41[医药卫生—肿瘤]