依赖于NRF2的lncRNA-mRNA共表达网络的全基因组识别及其在非小细胞肺癌进展中的作用

Genome-wide Identification of the lncRNA-mRNA Co-expression Network Dependent on NRF2 and its Role in the Progression of Non-small Cell Lung Cancer

目的研究长链非编码RNA(lncRNA)在mRNA和蛋白质水平上调节肺癌的发病和进展。方法从TCGA数据库下载486例非小细胞肺癌(NSCLC)及50例正常组织样本的RNA-seq数据,通过GEO数据库提取NRF2依赖性转录组以及3组NRF2-KD肺癌A549细胞系和3组用于免疫沉淀的对照细胞和NRF2激活下的NRF2-KD和A549细胞;利用ChIP-seq数据寻找上游10 kb和下游3 kb之间的启动子相关的峰值基因;使用TopHat2改进差异基因表达,再利用edgeR软件标准识别不同表达的lncRNA;对DE lincRNA和NRF2分别进行功能富集分析(GO、KEGG、Reactome途径);使用Cytoscape3.7.2对TCGA中550例NSCLC样本进行lncRNA-mRNA共表达分析;最后对上调和下调基因进行Kaplan-Meier生存分析。结果RNA-seq和H3K27ac ChIP-seq数据均来自于NRF2抑制和对照A549细胞;在含有NRF2 siRNA(NRF2-KD)的NRF2抑制A549样品中,共鉴定出3006个NRF2-DNA结合峰,依赖于NRF2的H3K27ac沉积峰在转录起始位点(TSS)附近的10 kb区域显示出较高的比例;NRF2-KD与A549细胞中共发现742个DE lncRNA表达差异(404个上调和368个下调),DE lncRNA的基因本体富集分析表明它们可能在炎症和免疫防御中发挥作用;通过依赖于NRF2的DE mRNA和H3K27ac结合的mRNA的重叠基因富集途径确定了增强子调节的18个DE lncRNA,并识别相关mRNA和两者关系的通路,在功能上与DNA修复、细胞周期和DNA复制相关;在18个lncRNA中,发现LINC00488上调与肺癌预后不良有关。结论lncRNA LINC00488的表达依赖于NRF2的结合,并且LINC00488的上调与肺癌患者的不良预后有关,其可以作为治疗肺癌新的预后生物标志物和靶点。

Objective To study the onset and progression of lung cancer by long-chain noncoding RNA(lncRNA)at the mRNA and protein levels.Methods RNA-seq data of 486 non-small cell lung cancer(NSCLC)and 50 normal tissue samples were downloaded from the TCGA database.The NRF2-dependent transcriptome and three groups of NRF2-KD lung cancer A549 cell lines and three groups of control cells for immunoprecipitation and NRF2-KD and A549 cells activated by NRF2 were extracted from the GEO database.ChIP-seq data were used to find promoter-related peak genes between upstream 10 kb and downstream 3 kb.TopHat2 was used to improve the differential gene expression,and the edgeR software standard was used to identify different expressed lncRNAs.Functional enrichment analysis of DE lincRNA and NRF2(GO,KEGG,Reactome pathway)Cytoscape 3.7.2 was used to analyze lncRNA-mRNA co-expression in 550 NSCLC samples from TCGA.Finally,Kaplan-Meier survival analysis was performed on up-regulated and down-regulated genes.Results Both RNA-seq and H3K27ac ChIP-seq data were derived from NRF2 inhibition and control A549 cells.A total of 3006 NRF2-DNA binding peaks were identified in NRF2 siRNA(NRF2-KD)-containing NRF2-suppressed A549 samples.The NRF2-dependent H3K27 ac deposition peak showed a high proportion in the 10 kb region near the transcription start site(TSS).A total of 742 DE lncRNA expression differences(404 up-regulated and 368 down-regulated)were found between NRF2-KD and A549 cells.Gene ontology enrichment analysis of DE lncRNAs showed that they might play a role in inflammation and immune defense.Through the overlapping gene enrichment pathway of NRF2-dependent DE mRNA and H3K27ac-binding mRNA,18 enhancer-regulated DE lncRNAs were identified,and related mRNAs and pathways were identified,which were functionally related to DNA repair,cell cycle and DNA replication.Among the 18 lncRNAs,up-regulation of LINC00488 was associated with poor prognosis of lung cancer.Conclusion The expression of lncRNA LINC00488 depends on the binding of NRF2,and the up-regulation of LINC00488 is related to the poor prognosis of lung cancer patients,which can be used as a new prognostic biomarker and target for the treatment of lung cancer.