Identification of aberrantly methylated differentially expressed genes in papillary thyroid carcinoma using integrated bioinformatic analysis
Authors: Li Qingxian , Cai Weiyi , Chen Jianhong , Ning Jie 
Published: April 2021

Abstract
Background: DNA methylation has been reported as one of the most critical epigenetic aberrations during the tumorigenesis and development of papillary thyroid carcinoma (PTC). Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified.Results: In this study, the comparisons between PTC and NT revealed 4995 methylated probes and 1446 differentially expressed transcripts cross-validated by The Cancer Genome Atlas (TCGA) database. The integrative analysis between DNA methylation and gene expression revealed 123 and 29 genes with hypomethylation/overexpression and hypermethylation/downexpression correlation, respectively. The DNA methylation pattern of seven selected CpGs (A: UNC80-cg04507925; B: TPO-cg09757588; C: LHX8-cg11842415; D: DLG2-cg16986720; E: FOXJ1-cg20373432; F: PALM2-cg21204870; G: IPCEF1-cg24635109, of which the candidate promoter CpG sites were preliminarily identified with the least absolute shrinkage and selection operator (LASSO) regression analysis. Then, the risk prognosis model was constructed by stepwise regression analysis. Furthermore, the receiver operating characteristic (ROC) and nomogram based on the verified independent prognostic factors was established for the prognostic prediction showed that it was able to predict 3-, 5-, and 7-year survival accurately. Kaplan-Meier survival estimate demonstrated that low DLG2 expression and DLG2-cg16986720 hypermethylation were independent biomarkers for OS. From the comprehensive meta-analysis, the combined Standardised Mean Difference (SMD) of DLG2 was 0.94 with 95% CI of (0.46,1.43), indicating that less DLG2 was expressed in the PTC tissue than in the normal tissue (P<0.05). Bisulfite sequencing PCR also showed that DLG2 methylation was higher in tumor group than in normal group. Components of immune microenvironment were analyzed using TIMER, and the correlation between immune cells and DLG2 was found to be distinct across cancer types. Based on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, DLG2 was implicated in pathways involved in immunity, metabolism, cancer, and infectious diseases. PCT patients with DLG2-cg16986720 hypermethylation showed significantly short survival rates in progression- free survival concomitant with reduced infiltration of myeloid dendritic cells.Conclusions: The current study validated that DLG2 was lowly expressed in PTC. More importantly, DLG2 hypermethylation might function as a latent tumor biomarker in the prognosis prediction for PTC. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of PTC. DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC. In addition to the promoter region, gene body and 3’UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.