CDH2 3'UTR Cloning and Mutation

The CDH2 3′-UTR was amplified by PCR from genomic DNA isolated from human blood. The pMIR-CDH2-3′UTR construct was digested with SpeI and MluI, and the generated fragment was inserted into the SpeI-MluI sites of the pMIR-REPORT miRNA Expression Reporter Vector (Applied Biosystems, Carlsbad, CA, USA). Three miR-218 binding sites in the CDH2 3′-UTR were predicted using miRSystem [29] (link), and these sites were located at 2,671–2,691 bp, 2,740–2,760 bp, and 3,571–3,591 bp relative to the transcription start site. Mutations were made in the miR-218 binding sites in the CDH2 3′-UTR using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol.