Pmirtarget 3 utr assay vector

The 3′UTR of BiP containing the putative miR-30a binding site was cloned into the pMirTarget 3′UTR assay vector (SC216266, OriGene, Rockville, MD, USA). YD38 and SCC25 cells were transfected with pBiP-3′UTR plasmid using Lipofectamine 2000 transfection reagent (11668019, Invitrogen, Carlsbad, CA, USA). The activity of luciferase was measured using a luciferase assay kit (16184, Thermo Fisher, Pittsburgh, PA, USA) according to the manufacturer’s instructions. The red fluorescence intensity was used to normalize firefly luciferase activity.

The Sp1 3′-UTR sequence that contained a predicted miR-145-5p binding site was constructed into a pMirTarget 3′-UTR assay vector (Origene Technologies, Inc., Rockville, MD, USA) after digesting with the restrict enzymes of EcoRI and XbaI. The mutant Sp1 3,’-UTR vector was constructed by deleting the predicted miR-145-5p binding site with a site-direct mutagenesis method using PCR synthesis of the wild type vector with mutant introducing primers followed by the digestion of the DpnI enzyme to remove the wild type sequences. The primer sequences for the constructions are provided in Table S2. Co-transfection of the miR-145-5p mimic or inhibitor with the wild type or mutant luciferase reporter constructs was performed by the TransIT^TM X2 transfection reagent. After being incubated for 48 h, the firefly luciferase activities were measured by the Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) and the firefly luciferase activity data were normalized by RFP signals after analyzing with a flow cytometer (BD FACSCalibur™ System, BD Biosciences, Franklin Lakes, NJ, USA.