Background: m6A RNA methylation modification plays an important role in the occurrence and progression of lung cancer and regulates tumor immunity. Current studies mostly focus on the differential expression of some specific m6A effectors and infiltrating immune cell. m6A methylation modification is the result of mutual adjustment and balance between effectors, and changes in the expression of one or two effectors are far from enough to reflect the panorama of m6A methylation. The role of m6A in the immune microenvironment of lung adenocarcinoma (LUAD) is still poorly understood. The aim of this study is to investigate the effect of different m6A modification patterns in immune microenvironment of LUAD.
Methods: LUAD data was obtained from The Cancer Genome Atlas (TCGA), University of California Santa Cruz Xena (UCSC Xena) and Gene Expression Omnibus (GEO) databases. Gene mutation, differential expression and survival analysis were performed for 24 m6A effectors. The m6A modification pattern was constructed by unsupervised clustering method, and the m6A clusters survival analysis, gene set variation analysis, immune score and immune cell infiltration analysis were performed. The association between LRPPRC protein expression levels and infiltration of CD8+ cytotoxic T lymphocytes and CD68+ macrophages in the tumor microenvironment was validated by immunohistochemistry in LUAD tissue microarray with 68 cases.
Results: The mutations of m6A effector were found in 150 of 567 LUAD cases with a frequency of 26.46%. 6 readers and 3 writers were significantly up regulated in LUAD tissues compared with normal tissues. IGF2BP1 and HNRNPC are the independent risk factors for prognosis of LUAD. Abundant cross-talks among writers, erasers and readers were demonstrated. Three m6A modification patterns with different immune cell infiltration characteristics and clinical prognosis were established. Among m6A effectors, LRPPRC was found to be inversely associated with the infiltration of CD8+ cytotoxic T lymphocytes and CD68+ macrophages, and was validated in 68 LUAD tissues.
Conclusions: m6A modification patterns play non-negligible roles in regulating the immune microenvironment. LRPPRC has potential to be a new biomarker for checkpoint inhibitor immunotherapy.
【中文题目：肺腺癌中m6A RNA甲基化修饰特征 与免疫微环境相关性分析】 【中文摘要：背景与目的 m6A RNA甲基化修饰在肺癌的发生与进展中起着重要作用，可以调节肿瘤免疫进而影响疾病预后。目前很多集中在某些特定的m6A效应器的差异表达及对肿瘤免疫细胞浸润的影响，但单个效应器表达的变化远远不足以反映m6A修饰特征的全貌，且关于m6A修饰对肺腺癌免疫微环境影响的研究仍较少。本研究拟探讨不同m6A修饰模式对肺腺癌中免疫微环境的影响。方法 从癌症基因组图谱数据库（The Cancer Genome Atlas, TCGA）、加州大学圣克鲁兹分校泛癌全基因数据分析工具数据库（University of California Santa Cruz Xena, UCSC Xena）、基因表达综合数据库（Gene Expression Omnibus, GEO）获取肺腺癌相关数据信息。使用Maftools R分析肺腺癌队列中24个m6A效应器的基因突变，比较肺腺癌组织和正常组织中m6A效应器的表达差异，并通过Cox回归分析进行生存分析。通过Consensus Cluster Plus R非监督聚类的方法构建m6A修饰模式，进行m6A聚类生存分析、GSVA通路富集分析、免疫评分及免疫细胞浸润分析。在68例肺腺癌组织中，通过免疫组化分析LRPPRC蛋白表达水平与CD8、CD68的表达水平，验证LRPPRC与CD8+细胞毒性T细胞及巨噬细胞浸润的关系。结果 在567例肺腺癌样本中有150例发生了m6A效应器突变，频率为26.46%。与正常组织相比，肺腺癌组织中共有6个读取器和3个写入器的表达明显上调。IGF2BP1和HNRNPC是影响肺腺癌患者预后的独立危险因素，且各效应器之间也存在大量的相互作用。构建了3种具有不同免疫细胞浸润特征和临床预后的m6A修饰模式。发现LRPPRC的表达与包括杀伤性T细胞和巨噬细胞在内的多种免疫细胞的浸润呈负相关，并在68例肺腺癌组织中得到验证。结论 m6A 修饰对肺腺癌免疫微环境的调节起着重要作用，LRPPRC有可能作为预测抗PD1免疫治疗效果的潜在生物标记物。】 【中文关键词：肺腺癌；m6A修饰；免疫微环境；LRPPRC】.
Keywords: Immune microenvironment; LRPPRC; Lung adenocarcinoma; m6A modification.