Targeting DCLK1 by miRNA-137.

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Targeting DCLK1 by miRNA-137. Targeting DCLK1 by miRNA-137. A, schematic representation of the predicted miR-137 target site sequence within the 3′UTR of DCLK1. DCLK1 had two predicted target sites (target 1 and target 2). Five nucleotides within target 1 and two nucleotides within target 2 complementary to the seed sequence (the nucleotide 2-7 of miRNA) of miR-137 were mutated in the DCLK1-mutant plasmid. The number indicates the position of the nucleotides in the wild-type sequence of DCLK1 (NM_00195415). B, activity of luciferase gene linked to the 3′UTR of DCLK1. The pGL3 luciferase reporter plasmids with the wild-type (WT) or mutated 3′UTR of DCLK1 (mut) were transiently infected into SW480 cells along with 25 nmol/L negative control or miR-137 precursor. Cotransfected Renilla luciferase reporter was used for the normalization. Luciferase activities were measured after 48 hours. The data are mean and SD of separated transfections (n = 3, *, P < 0.05, **, P < 0.01). C, semiquantitative RT-PCR analysis of DCLK1 mRNA expressions of colon cancer cell line SW480. SW480 cells expressed DCLK1. D, suppression of endogenous DCLK1 protein. SW480 cells were transfected with 25 nmol/L negative control (N.C.) or miR-137 precursor, and DCLK1 protein level was analyzed by Western blotting after 48 hours. Masazumi Sakaguchi et al. Mol Cancer Res 2016;14:354-362 ©2016 by American Association for Cancer Research