m5C修饰调控非小细胞肺癌 EGFR-TKI耐药的机制研究

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) resistance is a big concern during the therapy for non-small-cell lung cancer (NSCLC) patients. Accumulating evidence suggests that epigenetic alterations play vital roles in the cancer drug resistance. As a new research frontier of epigenetics, the effect of RNA 5-methylcytosine (m5C) modification in the regulation of cancer drug resistance is still unclear. We previously found that expression of the methyltransferase of mRNA m5C modification, NSUN2, was significantly increased in gefitinib-resistant NSCLC cells; whereas RNA interference of NSUN2 and the m5C-binding protein ALYREF reversed the drug resistance. Based on these findings, we hypothesize that abnormal m5C modification on RNA plays an important role in EGFR-TKI resistance through the regulation of drug resistance-associated genes. To validate this hypothesis, we will determine the role of m5C modification in EGFR-TKI resistance and identify the targeted genes that contribute to EGFR-TKI resistance with multidisciplinary approaches of molecular and cell biology, epigenetics and bio-information. The accomplishment of this project will illuminate the relationship between targeted genes, EGFR-TKI resistance and the prognosis of NSCLC and provide a novel target against EGFR-TKI resistance and important insights into mechanisms of drug resistance in NSCLC.

EGFR-TKI耐药是非小细胞肺癌(NSCLC)治疗中面临的严峻问题。表观遗传学在肿瘤耐药中扮演重要角色，RNA 5-甲基胞嘧啶 (m5C) 修饰是表观遗传学研究的前沿领域和热点。迄今关于m5C在肿瘤耐药中的作用尚不清楚。本团队前期发现：吉非替尼耐药NSCLC细胞中mRNA m5C甲基转移酶NSUN2表达显著上调，而下调NSUN2和m5C结合蛋白ALYREF表达能逆转吉非替尼耐药。据此我们提出假说：RNA m5C修饰异常是诱导EGFR-TKI耐药的重要原因，m5C修饰可通过调控耐药基因mRNA加工代谢影响其表达，进而诱导耐药。为验证这一假说，我们拟从细胞、模式动物及临床水平，综合分子和细胞生物学、表观组学和生物信息学等学科交叉手段，探讨m5C修饰对EGFR-TKI耐药的影响，明确m5C修饰调控的靶基因及机制，阐明靶基因与耐药及NSCLC预后的关系，为逆转EGFR-TKI耐药提供新策略。

耐药是非小细胞肺癌（NSCLC）靶向治疗面临的主要难题。5-甲基胞嘧啶（m5C）修饰是一种广泛存在于RNA中的化学修饰，但其在肿瘤耐药中的作用尚不清楚。团队前期研究发现，吉非替尼耐药NSCLC细胞中mRNA m5C甲基转移酶NSUN2表达显著上调，而敲低NSUN2表达能逆转吉非替尼耐药。在此基础上和本基金的资助下，本课题进一步探讨了RNA m5C修饰调控NSCLC EGFR分子靶向抑制剂（EGFR-TKIs）耐药的分子机制。在临床水平上，与EGFR-TKIs敏感患者相比，NSUN2及RNA m5C阅读蛋白（YBX1）在EGFR-TKIs原发性耐药患者中高表达，并与NSCLC预后差、体内转移密切相关；体内外功能实验证明过表达NSUN2可促进NSCLC对EGFR-TKIs耐药，而敲低NSUN2或YBX1可逆转NSCLC对EGFR-TKIs的耐药性，证实NSUN2-m5C-YBX1信号轴可介导EGFR-TKIs耐药进展。同时，整合RNA-BisSeq和RNA-seq测序数据分析发现QOSX1为NSUN2-m5C-YBX1信号轴调控的关键下游靶基因；原发性耐药细胞中QSOX1 mRNA CDS上m5C位点受到NSUN2调控，YBX1可结合到QSOX1 mRNA CDS上m5C促进其mRNA翻译，使QSOX1蛋白水平升高。QSOX1在EGFR-TKIs原发性耐药患者中显著高表达，在体内外敲低QSOX1可抑制耐药细胞的增殖、迁移能力以及体内肿瘤生长及转移能力。上述结果表明，异常RNA m5C高甲基化可通过NSUN2-YBX1-QSOX1信号轴促进NSCLC EGFR-TKIs原发性耐药进展，本研究可为逆转NSCLC EGFR-TKIs原发性耐药提供新的治疗策略。