Post-transcriptional regulation DPC4 gene by miR-190 in colorectal cancer cells.

Abstract

The objective of this study is to elucidate the regulation of the DPC4 gene by miR-190 in colorectal cancer (CRC) cells. The present study was undertaken to determine whether the DPC4 gene is a target gene of miRNA-190, identify target motifs and to elucidate the mechanism of regulation of DPC4 by miRNA-190. MiR-190 and DPC4 expression were measured in five different CRC cell lines by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The regulation of DPC4 by miR-190 was evaluated by qRT-PCR, Western blotting, and luciferase reporter assays in the human CRC cell line HT-29 after treatment with miR-190 mimics and inhibitors. The DPC4 mRNA, miR-, and DPC4 protein expression levels were highest in LS174T cells while lowest in SW480 and SW620 cells. The DPC4/miR-190 ratio in the HT-29 cancer cell line was the largest. MiR-190 expression increased dramatically after treatment with miR-190 mimics and decreased significantly after treatment with miR-190 inhibitors. DPC4 protein expression decreased in the miR-190 mimics transfection group when compared to the negative control (N.C.) group and increased in the miR-190 inhibitor groups when compared to the inhibitor plus N.C. group. MiR-190 inhibits the relative luciferase activity of psiCHECK-2™ vector-3'UTR compared to the N.C. group, while miR-190 had no obvious effect on the relative luciferase activity of the psiCHECK-2™ vector-3'UTRmut and psiCHECK-2™ vector transfected cells. The DPC4 gene might be the target gene of miR-190, which may negatively regulate the DPC4 gene in human CRC cells by translational suppression rather than mRNA degradation.