Scrambled mirna

A human miRNA expression vector (pEZX-MR04) encoding either hsa-miR31 or a scrambled miRNA (both from Gene Copoeia, Rockville, MD) was transfected into DLD-1 cells by using Lipofectamine 2000 as a transfection system. Transfection was performed as per manufacturer’s recommendation using a 4:1 (transfection reagent to plasmid) ratio. This ratio had been demonstrated to achieve the highest transfection efficiency (data not shown). Successful transfection of the transgene and expression of miR-31 was monitored by TaqMan qRT-PCR. RNU44 served as a house-keeping gene.

We performed a miRNA target validation luciferase reporter assay using H9c2 cells. In brief, cells were transfected with lentiviruses either GFP-tagged miR-18a-5p (cat# RmiR6078) or scrambled miRNA (cat# CmiR0001), which were purchased from GeneCopoeia. We received custom-designed HIF-1α 3′ untranslated region (UTR) clones (WT 3′UTR: cat# RmiT050798; mutant 3′UTR: cat# CS-RmiT050798) from GeneCopoeia, Rockville, MD, United States. For luciferase reporter assay, we co-treated 1 μg of WT or mutant HIF-1α 3′UTR clones with either miR-18a-5p or GFP-scrambled overexpression for 48 h, and measured the relative firefly to Renilla luciferase activity using a Dual-Glow Luciferase Assay kit (cat# E2920, Promega Corp., Madison, WI, United States) and a GloMax-Multi + Detection System (Promega) following the manufacturer’s manual.