背景:N6-甲基腺苷(m6A)修饰对于调节RNA加工和表达至关重要,特别是在恶性肿瘤进展的情况下。然而,鼻咽癌(NPC)中m6A修饰的探索仍然非常有限。
方法:使用m6A斑点印迹测定法分析NPC中的RNAm6A水平。从公共数据库以及RT-qPCR和免疫组织化学分析NPC组织中甲基转移酶样14(METTL14)的表达水平。通过体外和体内功能测定探讨了METTL14表达对NPC增殖和转移的影响。使用m6A和基因表达谱微阵列数据筛选METTL14的靶向基因。使用放线菌素D处理和多聚体分析来检测ANKRD22的半衰期和翻译效率。流式细胞术,免疫荧光和免疫沉淀用于验证ANKRD22对NPC细胞脂质代谢的作用。METTL14、GINS3、POLE2、PLEK2和FERMT1基因启动子附近的H3K27AC信号的ChIP-qPCR分析。
结果:我们在NPC中发现了METTL14,与不良患者预后相关。体外和体内测定表明METTL14积极促进NPC细胞增殖和转移。METTL14在ANKRD22信使核糖核酸(mRNA)上催化m6A修饰,由读者IGF2BP2识别,导致增加的mRNA稳定性和更高的翻译效率。此外,ANKRD22,线粒体上的代谢相关蛋白,与SLC25A1相互作用以增强柠檬酸盐的运输,提高细胞内乙酰辅酶A含量。ANKRD22的这种双重影响促进了脂质代谢重编程和细胞脂质合成,同时通过提高细胞核中的表观遗传组蛋白乙酰化水平来上调与细胞周期(GINS3和POLE2)和细胞骨架(PLEK2和FERMT1)相关的基因的表达。有趣的是,我们的发现强调了在METTL14启动子附近ANKRD22介导的组蛋白H3赖氨酸27乙酰化(H3K27AC)信号升高,这有助于一个正反馈循环延续NPC的恶性进展。
结论:确定的METTL14-ANKRD22-SLC25A1轴是NPC的有希望的治疗靶点,这些分子也可以作为新的诊断生物标志物。
BACKGROUND: N6-methyladenosine (m6A) modification is essential for modulating RNA processing as well as expression, particularly in the context of malignant tumour progression. However, the exploration of m6A modification in nasopharyngeal carcinoma (NPC) remains very limited.
METHODS: RNA m6A levels were analysed in NPC using m6A dot blot assay. The expression level of methyltransferase-like 14 (
METTL14) within NPC tissues was analysed from public databases as well as RT-qPCR and immunohistochemistry. The influences on
METTL14 expression on NPC proliferation and metastasis were explored via in vitro as well as in vivo functional assays. Targeted genes of
METTL14 were screened using the m6A and gene expression profiling microarray data. Actinomycin D treatment and polysome analysis were used to detect the half-life and translational efficiency of ANKRD22. Flow cytometry, immunofluorescence and immunoprecipitation were used to validate the role of ANKRD22 on lipid metabolism in NPC cells. ChIP-qPCR analysis of H3K27AC signalling near the promoters of
METTL14, GINS3, POLE2, PLEK2 and FERMT1 genes.
RESULTS: We revealed
METTL14, in NPC, correlating with poor patient prognosis. In vitro and in vivo assays indicated METTL14 actively promoted NPC cells proliferation and metastasis. METTL14 catalysed m6A modification on ANKRD22 messenger ribonucleic acid (mRNA), recognized by the reader IGF2BP2, leading to increased mRNA stability and higher translational efficiency. Moreover, ANKRD22, a metabolism-related protein on mitochondria, interacted with SLC25A1 to enhance citrate transport, elevating intracellular acetyl-CoA content. This dual impact of ANKRD22 promoted lipid metabolism reprogramming and cellular lipid synthesis while upregulating the expression of genes associated with the cell cycle (GINS3 and POLE2) and the cytoskeleton (PLEK2 and FERMT1) through heightened epigenetic histone acetylation levels in the nucleus. Intriguingly, our findings highlighted elevated ANKRD22-mediated histone H3 lysine 27 acetylation (H3K27AC) signals near the METTL14 promoter, which contributes to a positive feedback loop perpetuating malignant progression in NPC.
CONCLUSIONS: The identified METTL14-ANKRD22-SLC25A1 axis emerges as a promising therapeutic target for NPC, and also these molecules may serve as novel diagnostic biomarkers.