异常的 m5C 高甲基化通过 NSUN2/YBX1/QSOX1 轴介导 EGFR 突变型非小细胞肺癌对吉非替尼的内在耐药性。

BACKGROUND: RNA 5-methylcytosine (mC) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA mC modification in tumor drug resistance remain unclear. METHODS: The correlation between RNA mC methylation, mC writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and mC methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. RESULTS: RNA mC hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and mC-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant mC modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through mC reader Y-box binding protein 1 (YBX1). CONCLUSIONS: Our study reveals a critical function of aberrant RNA mC modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

背景：RNA 5-甲基胞嘧啶（mC）修饰在各种肿瘤的发病机制中发挥着关键作用。然而，RNA mC 修饰在肿瘤耐药中的功能和分子机制仍不清楚。

方法：在非小细胞肺癌（NSCLC）细胞系和患者样本中，确定 RNA mC 甲基化、mC 写入器 NOP2/Sun RNA 甲基转移酶家族成员 2（NSUN2）与 EGFR-TKIs 耐药之间的相关性。通过体外和体内的功能获得和功能丧失实验研究 NSUN2 对 EGFR-TKIs 耐药的影响。进行 RNA 测序（RNA-seq）、RNA 亚硫酸氢盐测序（RNA-BisSeq）和 mC 修饰 RNA 免疫沉淀-qPCR（MeRIP-qPCR）以鉴定 NSUN2 参与 EGFR-TKIs 耐药的靶基因。此外，通过功能挽救和嘌呤霉素掺入实验研究 NSUN2 调节靶基因表达的调控机制。

结果：RNA mC 高甲基化和 NSUN2 与 EGFR-TKIs 的内在耐药显著相关。NSUN2 的过表达导致吉非替尼耐药和肿瘤复发，而 NSUN2 的遗传抑制导致肿瘤消退并克服了吉非替尼在体外和体内的内在耐药。整合的 RNA-seq 和 mC-BisSeq 分析鉴定出硫氧还蛋白 1（QSOX1）是异常 mC 修饰的潜在靶标。NSUN2 甲基化 QSOX1 编码序列区，通过 mC 阅读器 Y 盒结合蛋白 1（YBX1）导致 QSOX1 翻译增强。

结论：我们的研究揭示了异常 RNA mC 修饰通过 NSUN2-YBX1-QSOX1 轴在介导 EGFR 突变型 NSCLC 对吉非替尼的内在耐药中的关键作用。