Cancer-associated methylated lncRNAs in patients with bladder cancer.

Epigenetic modifications via DNA methylation and long non coding RNAs (lncRNAs) have been identified in bladder cancer (BC). However, DNA methylation of lncRNAs involved in BC has not been elucidated. Here, DNA immunoprecipitation-sequencing (MeDIP-seq) and RNA-sequencing (RNA-seq) were carried out using eight paired tumor and adjacent normal tissue samples from patients with BC. Differences in methylation patterns between tumors and controls were compared and the percentage of differentially methylated genes, including lncRNA genes, was calculated. RNA-seq data were subjected to gene ontology (GO), Kyoto encyclopedia of genes, and genomes (KEGG) analysis. The association between DNA methylation modification and lncRNA expression was determined by pairwise analysis of MeDIP-seq and RNA-seq data. The most enriched motifs in the promoter region, as well as the methylated density in the 3 kb region surrounding super-enhancers of lncRNA genes, were analyzed. A peak of 5mC methylation in the region 2 kb upstream of the transcription start site (TSS), with the lowest point in the TSS region, was observed. In total, 436 and 239 genes were identified to be hyper and hypomethylated, respectively, in BC tissue around the TSS region. RNA seq revealed differentially expressed lncRNAs between tumor and normal tissues, many of which were cancer-associated lncRNAs based on GO and KEGG pathway analysis. Combined MeDIP-seq and RNA-seq analysis revealed that expression of 23 lncRNAs were candidates of 5mC controlled genes. The possible link between 5mC modification and differential IncRNAs may relate to enrichment of 5mC reads in the region surrounding super-enhancers of lncRNA. Survival analysis indicated that the methylated lncRNA, LINC00574, was associated with shorter overall survival time in patients with BC (HR = 1.7, p-value = 0.035). Taken together, these findings indicate that IncRNAs genes are under control of DNA methylation. Methylated lncRNA genes, which are transcripted to LINC00574, may serve as biomarkers for BC prognosis.