Exploration of Tumor-Suppressive MicroRNAs Silenced by DNA Hypermethylation in Oral Cancer

TLDR

MicroRNAs are pivotal regulators of carcinogenesis and are deregulated in a tumor‑specific manner across many cancers. The study aimed to identify a microRNA signature specific to oral squamous cell carcinoma. Researchers profiled 148 miRNAs in 18 OSCC cell lines versus the RT7 control, then focused on four CpG‑island‑associated miRNAs (miR‑34b, miR‑137, miR‑193a, miR‑203) to assess methylation‑mediated silencing and reactivation with 5‑aza‑2′‑deoxycytidine. They found 54 miRNAs down‑regulated in OSCC, with miR‑137 and miR‑193a frequently hypermethylated and re‑expressible after demethylation; ectopic expression of these miRNAs inhibited OSCC cell growth by targeting CDK6 and E2F6, confirming them as epigenetically silenced tumor suppressors. Cancer Res 2008;68(7):2094–2105.

Abstract

Abstract In the last few years, microRNAs (miRNA) have started a revolution in molecular biology and emerged as key players in the carcinogenesis. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature that was specific for oral squamous cell carcinoma (OSCC), we first examined expression profiles of 148 miRNAs in a panel of 18 OSCC cell lines and the immortalized oral keratinocyte line RT7 as a control. Compared with RT7, the expression of 54 miRNAs (36.5%) was frequently down-regulated in OSCC lines (<0.5-fold expression, ≥66.7% of 18 lines). Among these 54 miRNAs, we further analyzed four of these miRNAs (i.e., miR-34b, miR-137, miR-193a, and miR-203), located around CpG islands, to identify tumor-suppressive miRNAs silenced through aberrant DNA methylation. The expression of those four genes was restored by treatment with 5-aza-2′-deoxycytidine in OSCC cells lacking their expression. In addition, expression levels of the four miRNAs were inversely correlated with their DNA methylation status in the OSCC lines. In primary tumors of OSCC with paired normal oral mucosa, down-regulation of miRNA expression through tumor-specific hypermethylation was more frequently observed for miR-137 and miR-193a than for miR-34b and miR-203. Moreover, the ectopic transfection of miR-137 or miR-193a into OSCC lines lacking their expressions significantly reduced cell growth, with down-regulation of the translation of cyclin-dependent kinase 6 or E2F transcription factor 6, respectively. Taken together, our results clearly show that miR-137 and miR-193a are tumor suppressor miRNAs epigenetically silenced during oral carcinogenesis. [Cancer Res 2008;68(7):2094–105]