DNA methylation-mediated silencing of miR-204 is a potential prognostic marker for papillary thyroid carcinoma

Abstract

Background: Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and its incidence has increased over the last few decades. The molecular mechanisms underlying PTC tumorigenesis and progression are still unclear.

Patients and methods: The microRNA (miRNA) expression patterns of PTC were revealed by miRNA microarray analysis and validated with The Cancer Genome Atlas (TCGA) data. Promoter DNA methylation rates of miR-204 were analyzed by Agena Methylation MassAR-RAY analysis and validated with TCGA data. The underlying molecular mechanisms of miR-204 involved in PTC were studied by bioinformatics analyses.

Results: A total of 181 differentially expressed miRNAs (89 downregulated and 92 upregulated miRNAs) between PTC and normal tissues were detected in this study. We identified miR-204 as one of the most significantly downregulated miRNAs in PTC. Downregulation of miR-204 was related to PTC extrathyroidal extension, high T-stage, lymph metastasis, BRAF V600E mutation, and aggressive tall cell variant. The Agena MassARRAY results indicated that 12 CpG sites located at the promoter of miR-204 were hypermethylated in PTC tissues compared to normal tissues. The promoter methylation rates of miR-204 in PTC were negatively correlated with the expression levels of miR-204 and its host gene TRPM3. Downregulated miR-204 expression was related to several important pathways and mechanisms involved in tumorigenesis and progression.

Conclusion: Promoter DNA methylation-silenced miR-204 could serve as a potential diagnostic biomarker of PTC. Downregulation of miR-204 may play an important role in PTC via its involvement in many tumor-related pathways. Novel target genes and putative mechanisms of miR-204 in PTC need to be further validated.

Keywords: bioinformatics; miRNA; papillary thyroid carcinoma; promoter DNA methylation.

Figure 1

Heatmap of the 181 dysregulated miRNAs. Notes: Hierarchical analysis of the 181 dysregulated miRNAs based on the comparison of their expression values in PTC and normal tissues. Data have been deposited in GEO (accession code GSE113629). Abbreviations: GEO, Gene Expression Omnibus; miRNAs, microRNAs; PTC, papillary thyroid carcinoma; T, tumor tissues; N, normal tissues.

Figure 2

Downregulated miR-204 expression in PTC tissues and cell lines. Notes: (A) miR-204 expression levels are downregulated in human PTC tissues compared to those in paired adjacent normal tissues. (B) miR-204 expression levels are downregulated in human PTC cell lines compare to that in normal cell line. *P<0.05. Abbreviation: PTC, papillary thyroid carcinoma.

Figure 3

Promoter DNA methylation analysis of miR-204. Notes: (A) Schematic representation of the location of miR-204 in chromosome 9. miR-204 is located within the introns of TRPM3 (located at 9q21). (B) CpG island prediction and primer design of the miR-204 promoter region. Two CpG islands around the miR-204 promoter were identified using MethPrimer. Regions 9 and 15 that completely cover the two CpG islands were selected for the amplification. Each numbered region represents a region that can be amplified.

Figure 4

Promoter DNA methylation analysis of miR-204 with TCGA data. Notes: (A) Correlation analysis between miR-204 expression levels and promoter methylation levels. The expression levels of miR-204 in tumor tissues were inversely correlated with the promoter DNA methylation rates. (B) Methylation levels of CpG sites from TCGA data. Three of the four CpG sites were hypermethylated in tumor tissues compared to normal tissues. One CpG site was hypomethylated (cg14223966). (C and D) qRT-PCR demonstrated increased expression of miR-204 in PTC cells after treatment with 5-Aza-dye for 48 and 98 hours compared to mock-treated cells. *P<0.05. Abbreviations: 5-Aza, 5-aza-2′-deoxycytidine; PTC, papillary thyroid carcinoma; qRT-PCR, quantitative real-time PCR.

Figure 5

miR-204 promoter methylation rates in multiple cancers. Notes: In 13 types of cancers, higher methylation rates were observed in cancer tissues than in normal tissues. *P<0.05, **P<0.005.

Figure 6

GO and KEGG enrichment analyses of miR-204-related DEGs. Notes: (A) Significantly enriched GO terms of DEGs in PTC based on their functions. GO term classifications include biological process, cellular component, and molecular function. (B) Enriched KEGG pathways of miR-204-related DEGs. KEGG terms, gene numbers, P-values, and fold enrichment scores are presented in the pathway enrichment analysis. (C) Downregulation of miR-204-related pathway enrichment analysis by GSEA. The top enriched pathways showed significant enrichment in PTC tissues with lower miR-204 expression levels compared to tissues with higher miR-204 expression levels. Abbreviations: DEG, differentially expressed gene; GO, gene ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; PTC, papillary thyroid carcinoma.