基于DNA甲基化和长链非编码RNA鉴定肺鳞状细胞癌的预后生物标志物

Identification of prognostic biomarkers for lung squamous cell carcinoma based on DNA methylation and long non-coding RNAs

目的寻找与肺鳞状细胞癌(LUSC)预后相关的受甲基化调控的长链非编码RNA(lncRNA)。方法从癌症基因组图谱(TCGA)数据库下载502例LUSC患者的基因表达、甲基化和临床数据,进行甲基化和lncRNA表达的差异分析。筛选出位于差异甲基化区域(DMRs)的lncRNA,并进行生存分析和临床相关性分析。构建竞争性内源性RNA网络并对网络中的靶基因进行通路富集分析。用实时荧光定量聚合酶链式反应(qPCR)在14例LUSC术后组织样本中验证lncRNA的表达。结果共鉴定出40680个差异甲基化位点(dm-CpGs)和14418个DMRs。dm-CpGs大多数位于基因体,转录起始位点上游200、1500 bp和5’非翻译区(UTR)。37个差异表达的lncRNAs(DElncRNAs)位于DMRs中。其中14个DElncRNAs与DNA甲基化水平呈负相关。生存分析显示,3个DElncRNAs(TBX5-AS1、SFTA3、MAGI2-AS3)低表达组的总生存率(OS)均高于高表达组[5年OS(TBX5-AS1):50.8%比45.3%,风险比(HR)=1.39,P<0.05;5年OS(SFTA3):54.4%比41.8%,HR=1.37,P<0.05;5年OS(MAGI2-AS3):52.6%比43.3%,HR=1.44,P<0.05]。多因素Cox回归分析显示,由这3个lncRNA所构建的预后模型可作为LUSC总生存的独立预测因素(HR=2.746,95%可信区间:1.380~5.466,Z=2.88,P<0.01)。SFTA3在女性LUSC患者中表达高于男性[0.964(0.909,1.216)比0.672(0.393,1.014),W=26701,P<0.01]。TBX5-AS1在>60岁患者中表达高于≤60岁患者[0.720(0.411,1.034)比0.596(0.298,0.907),W=22152,P<0.05],在女性患者中表达高于男性[0.746(0.453,1.078)比0.637(0.370,1.002),W=19635,P<0.05],在Ⅳ期患者中表达高于Ⅰ+Ⅱ+Ⅲ期患者[0.964(0.909,1.216)比0.672(0.393,1.014),W=740,P<0.05]。通路富集分析显示MAGI2-AS3的靶基因主要富集PI3K-Akt、MAPK和Rap1等信号通路。qPCR结果显示,临床LUSC样本中TBX5-AS1、SFTA3、MAGI2-AS3在肿瘤中表达均低于正常组织[TBX5-AS1:0.006(0.001,0.013)比0.019(0.012,0.044),W=154,P<0.01;SFTA3:0.003(0.001,0.004)比0.022(0.015,0.030),W=187,P<0.01;MAGI2-AS3:0.030(0.009,0.052)比0.148(0.094,0.217),W=162,P<0.01]。结论lncRNA(SFTA3、MAGI2-AS3和TBX5-AS1)的表达与LUSC预后密切相关,在LUSC中表达下调且受DNA甲基化调控。

ObjectiveTo seek for methylation-regulated long non-coding RNAs(lncRNAs)associated with prognosis of lung squamous cell carcinoma(LUSC).MethodsThe transcriptome profiling,methylation data,and clinical information of 502 LUSC patients were retrieved from The Cancer Genome Atlas(TCGA)database.A genome-wide analysis of differential DNA methylation and lncRNA expression was performed.lncRNAs located in differentially methylated regions(DMRs)were screened out.Survival analysis and clinical correlation analysis were performed.A ceRNA network was constructed and pathway enrichment analysis was conducted based on target mRNAs in the netwrok.The expression level of lncRNAs in post-operative tissue of 14 cases of LUSC was validated by real-time quantitative polymerase chain reaction(qPCR).ResultsA total of 40680 differentially methylated CpGs(dm-CpGs)and 14418 DMRs across the whole genome were identified.Most dm-CpGs located in body regions,TSS1500,5’UTR,and TSS200 regions.A total of 37 differentially expressed lncRNAs(DElncRNAs)were found in DMRs,and 14 DElncRNAs of them were negatively associated with the DNA methylation levels.Survival analysis showed that overall survival(OS)was higher in low expression of the 3 DElncRNAs(TBX5-AS1,SFTA3,MAGI2-AS3)group than that in high expression group[five-year OS(TBX5-AS1):50.8%vs.45.3%,hazard ratio(HR)=1.39,P<0.05;five-year OS(SFTA3):54.4%vs.41.8%,HR=1.37,P<0.05;five-year OS(MAGI2-AS3):52.6%vs.43.3%,HR=1.44,P<0.05].Multivariate Cox regression analysis showed that a prognostic signature established with the 3 lncRNAs could be identified as an independent prognostic factor for LUSC(HR=2.746,95%confidence interval:1.380-5.466,P<0.01).SFTA3 expression was higher in female LUSC patients than in male[0.964(0.909,1.216)vs.0.672(0.393,1.014),W=26701,P<0.01].The expression of TBX5-AS1 in patients>60 years old was higher than that in patients≤60 years old[0.720(0.411,1.034)vs.0.596(0.298,0.907),W=22152,P<0.05].The TBX5-AS1 expression in female patients was higher than that in male[0.746(0.453,1.078)vs.0.637(0.370,1.002),W=19635,P<0.05],that in stageⅣpatients was higher than that in early stage[0.964(0.909,1.216)vs.0.672(0.393,1.014),W=740,P<0.05].The result of pathway enrichment analysis showed that the target mRNAs of MAGI2-AS3 were mainly involved in PI3K-Akt,MAPK and Rap1 signaling pathway.The result of qPCR showed that the expression levels of TBX5-AS1,SFTA3 and MAGI2-AS3 in clinical LUSC samples were lower in tumor than in normal tissue[TBX5-AS1:0.006(0.001,0.013)vs.0.019(0.012,0.044),W=154,P<0.01;SFTA3:0.003(0.001,0.004)vs.0.022(0.015,0.030),W=187,P<0.01;MAGI2-AS3:0.030(0.009,0.052)vs.0.148(0.094,0.217),W=162,P<0.01].ConclusionExpression level of three lncRNAs(SFTA3,MAGI2-AS3,and TBX5-AS1)were associated with prognosis of LUSC.Down-regulation of the three lncRNAs in LUSC were regulated by methylation.