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Methylation Mediated Silencing of MicroRNA-1 Gene and Its Role in Hepatocellular Carcinogenesis

Departments of ¹ Molecular and Cellular Biochemistry, ² Pathology, and ³ Molecular Virology, Immunology and Medical Genetics, ⁴ College of Pharmacy, and ⁵ Comprehensive Cancer Center, Ohio State University, Columbus, Ohio

Requests for reprints: Samson T. Jacob, The Ohio State University, 420 West 12th Avenue, 646B TMRF Building, Columbus, OH 43210. Phone: 614-688-5494; Fax: 614-688-5600; E-mail: Samson.Jacob{at}osumc.edu or Kalpana Ghoshal, The Ohio State University, 420 West 12th Avenue, 646C TMRF Building, Columbus, OH 43210. Phone: 614-292-8865; Fax: 614-688-5600; E-mail: Kalpana.Ghoshal{at}osumc.edu.

Key Words: microRNA • miR-1 • microarray • hepatocellular carcinoma • methylation mediated suppression • MET • FoxP1 • HDAC4

MicroRNAs (miR) are a class of small (21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1–/– HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3'-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]