Abstract A16: MiR-22 down-regulation via epigenetic control in oral cancer cells

Abstract

Abstract The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. DNA methylation in the CpG islands plays a crucial role in the down-regulation of tumor suppressor genes, including miRNAs. MiR-22 has been shown to be deregulated in several cancer types, including breast cancer and multiple myeloma. Using real-time RT-PCR analysis, we also found that miR22 was down-regulated in the majority of tested oral cancer cell lines and 70% of the clinical specimens when compared with their normal counterparts. Consistent with the notion that DNA methylation might play a role in the gene silencing, we did detect a putative CpG island (~ 1.2 kb in length) located upstream from the putative miR-22 transcription start site (+1) using two independent CpG island prediction softwares. Two oral cancer lines with low miR−22 expression were treated for 3–5 days with the indicated dose of 5′-azaC together with or without trichostatin A. Total RNA was isolated from the treated cells for real-time quantitative PCR analysis of miR-22 expression. The expression of miR-22 was significantly induced by either inhibitor. Combined treatment further enhanced the expression, suggesting the involvement of epigenetic control in the decreased expression of miR-22 in oral cancer cells. We first divided the putative CpG island into 5 regions and designed nested PCR primers for bisulfate sequencing PCR. We found that only regions 3 to 5 in the putative CpG island were subjected to be influenced by methylation in certain oral cancer lines. Since MeCP2 selectively binds to methylated CpG dinucleotides, chromatin immunoprecipition together with qPCR will be used to examine if MeCP2 would have increased binding to the identified methylated CpG sites. Second, quantitative methylation specific PCR (qMSP) will be used to measure the methylation levels of miR-22 genes in oral cancer tissues and their normal control using SYBR Green–based detection technology. Third, in vitro methylation and promoter-driven luciferase assay will be used to investigate whether DNA methylation plays a direct role in regulating miR-22 promoter activity and whether CpG island deletion affects the relative luciferase activity. Together, this study would implicate that the expression of miR-22 is controlled by epigenetic control in oral cancer cells. However, the exact nature of miR-22 in oral carcinogenesis remains to be characterized. Citation Format: Li-Wha Wu. MiR-22 down-regulation via epigenetic control in oral cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A16.