682 Inactivation of T-Box 5 (Tbx5) Gene in Colon Cancer and Its Potential Application

Abstract

Background and Aims: microRNAs (miRNAs) are involved in the pathogenesis of multiple human cancers, including CRC. Dysregulation of global miRNA profiles, which often associates with loss of expression of certain miRNAs, is a common feature in CRC. Whereas CpG island hypermethylation has been proposed to be a potential mechanism for miRNA silencing, this field remains largely unexplored. In this study, we describe the epigenetic regulation of miR-137 and its contribution to colorectal carcinogenesis. Methods: We determined the methylation status of the miR-137 CpG island in a panel of six CRC cell lines and 409 colorectal tissues (21 normal colonic mucosae from healthy individuals (N-N), 160 primary CRC tissues with corresponding adjacent normal colonic mucosa (N-C) and 68 colonic adenomas) by bisulfite pyrosequencing. TaqMan RT-PCR and in situ hybridization (ISH) were used to analyze miR-137 expression. In Vitro functional analysis of miR-137 was performed using precursor-miRNA transfection. Downstream genetic targets of miR-137 were identified by gene expression microarray analysis in combination with in silico predictions. Western blotting and luciferase assays were performed to validate the miRNA:mRNA interactions. Results: Methylation of the miR-137 CpG island was frequent in CRC cell lines (100%), adenomas (82.3%) and CRCs (81.4%) but not in N-C (14.4%; p<0.0001 for CRC) and N-N (4.7%; p<0.0001 for CRC). When compared to miR-137 unmethylated tumors, methylated CRCs were more frequently associated with KRAS mutations (37% vs 14%; p= 0.046) and older age (66 vs 60 years; p= 0.024). ISH results revealed that miR-137 expression was restricted to the colonocytes in normal mucosa, and was absent in CRC tissues. TaqMan RT-PCR analysis showed an inverse correlation between methylation and expression both in CRC tissues and cell lines. Transfection of the miR-137 precursor into CRC cell lines significantly decreased the cell proliferation index (p=0.0029). Functional assays validated an interaction between miR-137 and LSD-1, which plays a crucial role in the maintenance of DNAmethylation.Conclusions: Our data suggest that miR-137 acts as a tumor suppressor in the colon, and is frequently silenced by promoter hypermethylation. Frequent methylation found in adenomatous polyps suggests it is an early event in colorectal carcinogenesis. The interaction between miR-137 and LSD-1 suggests a mechanistic explanation for these observations and highlights the potential significance of this tumor-suppressive miRNA in epigenetic processes in colorectal tumorigenesis.