M6A修饰EGLN3、FOSL2参与鼻咽癌辐射抗拒

M6A-mediated EGLN3 and FOSL2 enhance radioresistance in nasopharyngeal carcinoma

目的基于芯片数据并通过细胞实验验证鼻咽癌受m6A甲基化调控的关键放射抗拒基因。方法从GEO数据库分别下载鼻咽癌放射抗拒基因与鼻咽癌m6A调控基因以及鼻咽癌基因mRNA表达谱芯片数据,使用R软件进行差异基因的筛选与统计学分析,采用生物信息学方法对这些基因进行生物学过程、信号通路及互作网络分析。分别通过敲低m6A调控因子、放射抵抗株构建,以实时反转录PCR(qRT-PCR)和蛋白质印迹法对上述鼻咽癌m6A差异放射抗拒基因进行筛选验证。甲基化RNA免疫共沉淀后实时定量PCR(MeRIP-qPCR)验证筛选基因的m6A修饰及与甲基转移酶3(METTL3)直接结合的能力。在鼻咽癌细胞中转染筛选基因的siRNA,电离辐射处理后,用CCK-8、EdU法检测细胞增殖,流式细胞术检测细胞凋亡和细胞周期,克隆形成实验检测放射敏感性。通过配对t检验,比较电离辐射处理后对照组与EGLN3敲减组Fe^(2+)丰度及脂质过氧化程度的差异趋势。结果芯片数据GSE48501与GSE200792、GSE53819交集得到6个差异基因,分别是EGLN3、FOSL2、ADM、JUN、VEGFA、PRDM1。经TNMplot、KMplot等生信数据平台进一步筛选得到目标基因EGLN3、FOSL2。目标基因mRNA直接与METTL3结合并受到其介导的m6A修饰。目标基因在电离辐射后的亲代细胞中呈现剂量、时间梯度上调;在放射抵抗细胞中也显著上调。沉默EGLN3/FOSL2后的鼻咽癌细胞经照射处理后,细胞增殖活性下降更多,与未下调者的放射增敏比的差异有统计学意义。沉默EGLN3后的鼻咽癌细胞经照射处理后,显著下调谷胱甘肽过氧化物酶4(GPX4)表达,并进一步增加Fe^(2+)丰度及脂质过氧化程度,通过诱发铁死亡发挥放疗增敏作用。结论EGLN3、FOSL2在鼻咽癌中通过METTL3介导的m6A甲基化修饰发挥放射抵抗作用;下调EGLN3并联合电离辐射可以增加鼻咽癌细胞内Fe^(2+)丰度、脂质过氧化程度并降低GPX4表达,以铁死亡途径增鼻咽癌放射敏感性。

Objective To screen and verify the key radioresistance genes regulated by m6A methylation in nasopharyngeal carcinoma(NPC)based on the chip data and cell experiments.Methods The microarray data of NPC radioresistance genes,m6A regulated genes and mRNA expression profiles of NPC genes were downloaded from Gene Expression Omnibus(GEO)database.The differential genes were screened and statistically analyzed by R software.The biological processes,signal pathways and interaction networks of these genes were analyzed by bioinformatics.The m6A regulatory factors were knocked down and the radioresistant strains were constructed.The above m6A differential radioresistant genes of NPC were screened and verified by real-time reverse transcription PCR(qRT-PCR)and Western blot.The m6A modification of screened genes and their direct binding ability with methyltransferase 3(METTL3)were verified by methylated RNA immunoprecipitation qPCR(MeRIP-qPCR).The siRNA of selected genes was transfected into NPC cells,and after treatment with ionizing radiation,cell proliferation was detected by CCK-8 assay and EdU,apoptosis and cell cycle were detected by flow cytometry,and radiosensitivity was detected by clone formation assay.The trend of differences in the abundance of Fe^(2+)and lipid peroxidation between the control and EGLN3 knockdown groups after ionizing radiation treatment was compared by paired t-test.Results Chip data GSE48501 intersected with GSE200792 and GSE53819 to obtain 6 differential genes,including EGLN3,FOSL2,ADM,JUN,VEGFA and PRDM1.The target genes of EGLN3 and FOSL2 were further screened by TNMplot and KMplot,etc.The mRNA of the target genes directly bound to METTL3 and were subjected to its mediated modification of m6A.The target genes were up-regulated in the parental cells after irradiation in a dose and time gradient manner,which were also significantly up-regulated in radioresistant cells.After EGLN3 and FOSL2 were down regulated,the proliferation activity of NPC cells was more significantly decreased after irradiation,and the radiosensitization ratio was statistically significant compared with that of NPC cells without EGLN3 and FOSL2 down-regulation.After irradiation,EGLN3 down-regulated NPC cells significantly down-regulated glutathione peroxidase 4(GPX4)expression,increased the abundance of Fe^(2+)and lipid peroxidation,which played a role in radiosensitization by inducing ferroptosis.Conclusions EGLN3 and FOSL2 play a role in radioresistance in NPC through METTL3 mediated m6A methylation.Down-regulation of EGLN3 combined with ionizing radiation can increase the intracellular Fe^(2+)abundance and lipid peroxidation and down-reuglate the expression of GPX4 in NPC cells,which can enhance radiosensitization for NPC radiotherapy by the ferroptosis pathway.