YTHDF2抑制NSCLC发生、发展的相关分子机制

Molecular Mechanism of YTHDF2 Inhibiting the Occurrence and Progression of NSCLC

摘要: 背景与目的：非小细胞肺癌(non-small cell lung cancer, NSCLC)是目前全球发病率最高和死亡人数最多的恶性肿瘤，而其具体的发病机制仍然未知。相关文献和既往实验研究发现m6A的阅读蛋白YTHDF2可能通过分子之间的相互作用抑制NSCLC的发生、发展。本研究拟通过对NSCLC患者肿瘤组织进行病理观察以及生信分析，探索NSCLC的发生发展机制，对未来产生更加有效的NSCLC治疗手段和改善NSCLC患者的预后提供参考。方法：回顾性收集2021年5月至2022年5月于青岛大学胸外科确诊肺癌并住院手术的64例NSCLC患者的肿瘤及癌旁组织(根据世界卫生组织形态学标准)。对上诉样本分别进行病理染色及生信分析。结果：64例标本的HE染色可见肿瘤组织中病理损伤明显，能明确采集样本的诊断并与癌旁组织区分。TUNEL染色可见：肿瘤组织细胞凋亡水平升高。qRT-PCR实验结果表明在肿瘤组织中YTHDF2和p14基因表达水平明显高于癌旁组织(P < 0.05)。且Spearman等级相关性分析YTHDF2与p14在NSCLC肿瘤组织中的相对表达水平呈正相关(rs = 0.561, P < 0.001)。Western Blot检测组织YTHDF2表达水平，结果同qRT-PCR结果一致，肿瘤组织中的YTHDF2表达上调(P < 0.001)。TET2生信分析显示TET2 mRNA上存在大量的m6A位点，且GEPIA数据库显示YTHDF2与TET2的表达存在正相关性。甲基化特异性PCR检测结果表明肿瘤组织的p14 DNA中明确存在高甲基化修饰。根据GEPIA数据库显示在NSCLC中YTHDF2和TET2表达呈正相关，且qRT-PCR实验发现YTHDF2和p14在NSCLC肿瘤组织中的相对表达水平存在正相关性。结论：在NSCLC中，m6A的阅读蛋白YTHDF2可能通过识别TET2 mRNA上的m6A位点增加TET2蛋白的表达，发挥其去甲基化作用使p14 DNA启动子高甲基化区域发生去甲基化，促进p14^ARF蛋白的表达，达到延缓NSCLC发生、发展和改善NSCLC患者预后的作用。

Abstract: Background and objective: Non-small cell lung cancer (NSCLC) is currently the most prevalent and deadly malignancy worldwide. However, the precise mechanisms underlying its pathogenesis remain largely unknown. Previous literature and experimental studies suggest that YTHDF2, an m6A reader protein, may inhibit the initiation and progression of NSCLC through molecular interactions. This study aims to explore the mechanisms involved in the development and progression of NSCLC by conducting pathological observations and bioinformatic analyses of tumor tissues from NSCLC patients. The findings are expected to contribute to the development of more effective treatment strategies for NSCLC and to improve the prognosis of NSCLC patients. Methods: We retrospectively collected tumor and adjacent normal tissues from 64 NSCLC patients who were diagnosed and underwent surgery between May 2021 and May 2022 in the Department of Thoracic Surgery at Qingdao University, following the World Health Organization (WHO) morphological criteria. Pathological staining and bioinformatic analyses were performed on the collected samples. Results: Hematoxylin and eosin staining of the 64 specimens revealed significant pathological damage within the tumor tissue, which could clearly diagnose the collected samples and distinguish them from the adjacent tissues. TUNEL staining showed that the level of apoptosis in tumor tissues was increased compared with adjacent tissues. The results of qRT-PCR showed that the expression levels of YTHDF2 and p14 genes in tumor tissues of NSCLC patients were significantly higher than those in adjacent tissues (P < 0.05). Spearman rank correlation analysis showed that the relative expression levels of YTHDF2 and p14 in NSCLC tumor tissues were positively correlated (rs = 0.561, P < 0.001). The expression level of YTHDF2 in tumor tissues was detected by Western Blot, and the results were consistent with the results of qRT-PCR. Compared with the adjacent tissues of NSCLC patients, the expression of YTHDF2 in tumor tissues was up-regulated (P < 0.001). The bioinformatics analysis of TET2 showed that there were a large number of m6A sites on TET2 mRNA, and GEPIA database showed that the expression of YTHDF2 was positively correlated with TET2 (R = 0.5 in lung adenocarcinoma and R = 0.48 in lung squamous cell carcinoma). The results of methylation-specific PCR showed that p14 DNA was clearly hypermethylated in NSCLC tumor tissues. According to GEPIA database, the expression of YTHDF2 and TET2 was positively correlated in NSCLC, and qRT-PCR experiment showed that the relative expression levels of YTHDF2 and p14 in NSCLC tumor tissues were positively correlated. Conclusion: These results suggest that in NSCLC, the m6A reading protein YTHDF2 may increase TET2 protein expression by recognizing the m6A site on TET2 mRNA, and exert its demethylation effect to demethylation of the hypermethylated region of p14 DNA promoter and promote p14^ARF protein expression. It can delay the occurrence and development of NSCLC and improve the prognosis of NSCLC patients.