Identification of prognostic biomarkers for lung squamous cell carcinoma based on DNA methyla-tion and long non-coding RNAs
Objective To seek for methylation-regulated long non-coding RNAs(lncRNAs)asso-ciated with prognosis of lung squamous cell carcinoma(LUSC).Methods The 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).Results A total of 40 680 differentially methylated CpGs(dm-CpGs)and 14 418 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,MAG12-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 re-gression 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=26 701,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=22 152,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=19 635,P＜0.05],that in stage Ⅳ patients was high-er 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].Conclusion Expression 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.
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