Identifying and Validating microRNA Targets

Luciferase reporter transfection and dual luciferase assays were performed following protocols established in our previous reports.^{26 (link),27 (link)} We used the computational target prediction algorithm miRanda (http://cbio.mskcc.org/microRNA2003/miranda.html) to identify mature gene transcripts that were potentially targeted by JAM-A at their 3′ UTRs. VCAN was then experimentally verified as a JAM-A target gene in hDPCs following the protocols of Wang et al.^{26 (link),27 (link)} Reporter assays used Light Switch 3′ UTR Go Clone Constructs (BioCat GmBH, Heidelberg, Germany) based on the 3′ UTR sequences of JAM-A and VCAN, which were co-transfected with 100 ng of VCAN reporter plasmid and either 10–40 nM miRNA-221–3p mimic or 40 nM control siRNA. After 24 h of incubation, 50 μl of Light Switch reagent (BioCat GmBH, Heidelberg, Germany) was added to each well, and luminescence was measured using a plate-reading luminometer (Tecan, Männedorf, Switzerland). RT–qPCR analysis was performed to quantify the VCAN transcription levels in DP cells at 1 day after incubation with the microRNA-221–3p mimic or inhibitor.