Altered Methylation at MicroRNA-Associated CpG Islands in Hereditary and Sporadic Carcinomas: A Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA)-Based Approach

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that contribute to tumorigenesis by acting as oncogenes or tumor suppressor genes and may be important in the diagnosis, prognosis and treatment of cancer. Many miRNA genes have associated CpG islands, suggesting epigenetic regulation of their expression. Compared with sporadic cancers, the role of miRNAs in hereditary or familial cancer is poorly understood. We investigated 96 colorectal carcinomas, 58 gastric carcinomas and 41 endometrial carcinomas, occurring as part of inherited DNA mismatch repair (MMR) deficiency (Lynch syndrome), familial colorectal carcinoma without MMR gene mutations or sporadically. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assays were developed for 11 miRNA loci that were chosen because all could be epigenetically regulated through the associated CpG islands and some could additionally modulate the epigenome by putatively targeting the DNA methyltransferases or their antagonist retinoblastoma-like 2 (RBL2). Compared with the respective normal tissues, the predominant alteration in tumor tissues was increased methylation for the miRNAs 1-1, 124a-1, 124a-2, 124a-3, 148a, 152 and 18b; decreased methylation for 200a and 208a; and no major change for 373 and let-7a-3. The frequencies with which the individual miRNA loci were affected in tumors showed statistically significant differences relative to the tissue of origin (colorectal versus gastric versus endometrial), MMR proficiency versus deficiency and sporadic versus hereditary disease. In particular, hypermethylation at miR-148a and miR-152 was associated with microsatellite-unstable (as opposed to stable) tumors and hypermethylation at miR-18b with sporadic disease (as opposed to Lynch syndrome). Hypermethylation at miRNA loci correlated with hypermethylation at classic tumor suppressor promoters in the same tumors. Our results highlight the importance of epigenetic events in hereditary and sporadic cancers and suggest that MS-MLPA is an excellent choice for quantitative analysis of methylation in archival formalin-fixed, paraffin-embedded samples, which pose challenges to many other techniques commonly used for methylation studies.

DNA methylation silences miR-132 in prostate cancer

“…The tumor-specific promoter methylation of miR-148a, miR-18b and miR-34b/c in cancers other than prostate has already been described. 12,16,17 Recently, miR-542-3p was shown to initiate apoptosis in cancer cells. 18 miR-132's differential expression between normal and tumorous tissues indicates a context-dependent role as either an oncomiR or a tumor suppressor with upregulated expression in lung, 19 breast 20 and tongue carcinomas, 21 and downregulated expression in osteosarcoma 22 and gliomas.…”

Identification of subgroup-specific miRNA patterns by epigenetic profiling of sporadic and Lynch syndrome-associated colorectal and endometrial carcinoma

“…We opted for a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)-based approach, which allows for multiplex, quantitative analysis of methylation in archival formalin-fixed, paraffin-embedded (FFPE) specimens without the need of bisulfite conversion [16]. A representative electropherogram from MS-MLPA analysis is shown in Figure 2.…”

Cancer epigenomics: beyond genomics

“…Saito et al showed that mir-127 was suppressed by promoter hypermethylation and by a decrease in acetyl-H3 and methyl-K4H3 in cancer cells [56]. Genome-wide analysis of different cancer types has shown the influence of DNA methylation and histone modifications on the global regulation of miRNAs [57][58][59].…”