m6A修饰调控免疫检查点PD-L1表达及肺腺癌免疫抑制的研究

背景 
肺癌是临床上常见恶性肿瘤之一,肺腺癌（lung adenocarcinoma，LUAD）是肺癌最常见的组织亚型，约占肺恶性肿瘤的40%，其较差的预后是导致患者临床治疗失败和高死亡率的主要原因。近年来，免疫检查点阻断疗法在癌症的治疗中取得较大突破。m6A修饰在恶性肿瘤中的作用得到越来越多的证实，然而m6A修饰与肿瘤免疫微环境的关系尚不清楚。
方法 
运用MeRIP-seq检测30例肺腺癌临床样本组织中m6A修饰水平，通过GO富集和KEGG富集分析肺腺癌组织中m6A水平上调及下调的基因所涉及的生物学功能和信号通路。联合RNA-seq及蛋白质组分析m6A修饰在转录及蛋白合成中的作用。临床样本RNA-seq测序结合TCGA数据库中肺腺癌数据分析m6A识别蛋白YTHDF1在肺腺癌肿瘤微环境中的角色。同时利用多色免疫荧光技术检测m6A识别蛋白YTHDF1与CD8+ T细胞、树突状细胞、巨噬细胞空间定位之间的相关性。RNA-seq，蛋白质组及代谢组学分析YTHDF1在肺腺癌中的重要作用，运用GO，KEGG和GSEA富集分析YTHDF1缺失影响的下游信号。利用siRNA/shRNA对肺腺癌细胞进行转染沉默YTHDF1，进而检测YTHDF1对肺腺癌增殖、迁移、侵袭及凋亡等生物学功能的影响。构建小鼠肺腺癌皮下移植瘤模型，检测沉默YTHDF1后对肺腺癌肿瘤生长的影响。通过m6A-RIP-qPCR和RNA pull-down实验检测YTHDF1与PD-L1 mRNA相互作用方式。
结果 
MeRIP-seq, RNA-seq 和蛋白质组联合分析，肺腺癌中642个基因RNA未改变，但m6A修饰水平和蛋白表达均发生明显改变。GO富集分析显示，mRNA分解代谢过程的调节，RNA稳定性的调节，RNA定位，mRNA稳定性的调节和 RNA转运等生物学过程被富集。KEGG富集分析，蛋白酶体，RNA转运，RNA降解和错配修复被富集。通过30例临床样本RNA-seq数据，TCGA肺腺癌数据，TIMER数据库，TISIDB数据库以及组织芯片的多色免疫荧光结合分析YTHDF1与免疫细胞相关性，发现YTHDF1抑制肺腺癌中免疫细胞浸润，促进肺腺癌免疫抑制。联合shYTHDF1 RNA-seq与蛋白质组数据分析，YTHDF1缺失与甘油酯代谢，果糖和甘露糖代谢，甘氨酸、丝氨酸和苏氨酸代谢和核苷酸代谢等代谢途径相关。细胞功能实验发现沉默YTHDF1能够抑制人肺腺癌细胞增殖、迁移、侵袭和克隆形成能力，并且促进细胞凋亡。通过皮下移植瘤模型的建立，沉默YTHDF1明显抑制小鼠肺腺癌移植瘤的生长。WB实验发现YTHDF1沉默下调PD-L1蛋白表达。进一步机制研究发现YTHDF1通过m6A修饰位点与PD-L1的mRNA结合，增强PD-L1 mRNA的翻译，从而上调PD-L1蛋白表达。
结论 
m6A识别蛋白YTHDF1通过调节CD8+ T细胞的浸润在抗癌免疫中发挥关键作用。并且YTHDF1以m6A修饰依赖方式促进免疫检查点PD-L1 mRNA的翻译，增加PD-L1蛋白表达，以此促进肺腺癌免疫逃逸及恶性进展。这表明靶向YTHDF1可能是一种潜在的肿瘤免疫治疗新策略。

Background

Lung cancer is one of the most common malignant tumors in clinical practice. Lung adenocarcinoma (LUAD) is the most common tissue subtype of lung cancer, accounting for about 40% of lung malignancies. Its poor prognosis is the main cause of clinical treatment failure and high mortality of patients. Recently, immune checkpoint blocking has been a breakthrough in the treatment of cancer. The role of m6A modification in malignant tumors has been more and more confirmed, but the relationship between m6A modification and tumor microenvironment remains unclear.

Methods

MeRIP-seq was used to detect the m6A modification level in 30 LUAD clinical samples. GO enrichment and KEGG enrichment were used to analyze the biological functions and signaling pathways involved in the up-regulated and down-regulated m6A genes in LUAD tissues. Combined with RNA-seq and proteomics, the role of m6A modification in transcription and protein synthesis was analyzed. The role of m6A recognition protein YTHDF1 in the microenvironment of LUAD in clinical samples by RNA-seq combined with LUAD data in the TCGA database. Meanwhile, the correlation between the m6A recognition protein YTHDF1 and the spatial localization of CD8+ T cells, dendritic cells, and macrophages was detected by multiplex immunohistochemistry. RNA-seq, proteomics, and metabolomics were used to analyze the important role of YTHDF1 in LUAD. GO, KEGG, and GSEA enrichment was used to analyze the downstream signals of YTHDF1 influence. siRNA/shRNA was used to transfect LUAD cells, to detect the effects of YTHDF1 on the biological functions of LUAD, such as proliferation, migration, invasion, and apoptosis. A mouse LUAD subcutaneous graft model was established to detect the effect of silencing YTHDF1 on the growth of LUAD. The interaction between YTHDF1 and PD-L1 mRNA was detected by m6A-RIP-qPCR and RNA pull-down experiments.

Results

MeRIP-seq, RNA-seq, and proteome analysis showed that the RNA of 642 genes in LUAD did not change, but the modification level of m6A and protein expression were significantly changed. GO enrichment analysis showed that biological processes such as regulation of mRNA catabolism, regulation of RNA stability, RNA localization, regulation of mRNA stability, and RNA transport were enriched. KEGG enrichment analysis, proteasome, RNA transport, RNA degradation, and mismatch repair were enriched. The correlation between YTHDF1 and immune cells was analyzed by the combination of RNA-seq data, TCGA-LUAD data, TIMER database, TISIDB database, and tissue chip in 30 clinical samples. It was found that YTHDF1 inhibited the infiltration of immune cells in LUAD and promoted the immunosuppression of LUAD. In combination with shYTHDF1 RNA-seq and proteome data analysis, YTHDF1 deletion was associated with metabolic pathways such as triglyceride metabolism, fructose and mannose metabolism, glycine, serine, and threonine metabolism, and nucleotide metabolism. Function experiments showed that silencing YTHDF1 inhibited the proliferation, migration, invasion, and clone formation of lung adenocarcinoma cells, and promoted cell apoptosis. By establishing a subcutaneous graft model, silencing YTHDF1 significantly inhibited the growth of LUAD grafts in mice. WB experiment showed that YTHDF1 silencing down-regulated PD-L1 protein expression. Further mechanism study found that YTHDF1 binds to PD-L1 mRNA through the m6A modification site, enhancing the translation of PD-L1 mRNA and thereby increasing the expression of the PD-L1 protein.

Conclusion

The m6A recognition protein YTHDF1 plays a key role in anticancer immunity by regulating the infiltration of CD8+ T cells. Moreover, YTHDF1 promoted the translation of PD-L1 mRNA and increased the expression of PD-L1 protein in an m6A modification-dependent manner, thus promoting the immune escape and malignant progression of LUAD. This suggests that targeting YTHDF1 may be a potential new strategy for cancer immunotherapy.

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