Lipofectamine

The immortalized human bronchial airway epithelial cell line BEAS-2B S.6 [35] (link) was transfected with 30 nM mirVana mimics of hsa-miR-149-5p, hsa-miR-202-3p, hsa-miR-410 and the miR#1 negative control (lacking any mammalian target) using lipofectamine (Applied Biosystems). After 24 h, transfected cells were evaluated for microRNAs, IL-6 mRNA and IL-6 protein levels. For microRNA binding analysis, the papilloma virus–immortalized human bronchial epithelial cell line HBE [36] (link) was transfected with a 3′ UTR IL-6 gene target cloned upstream of a Firefly luciferase reporter in a dual luciferase reporter system (GeneCopoeia, Rockville, MD) using lipofectamine. HBE cells were co-transfected with the IL-6 3′ UTR clone and 40 nM microRNA mimics, followed by evaluation of relative luciferase activity after 24 h. For transfection-normalization across samples, the firefly luciferase is reported relative to an internal control, consisting of renilla luciferase driven by a CMV promoter.

The wild and mutation types of SP1 3'‐UTR luciferase vectors (0.6 μg/ml each) obtained from GeneCopoeia, Inc. (Rockville, MD, USA) were transfected into the cells with either miR7‐5p mimics (100 nM) or negative control using lipofectamine 3000 reagent; the preparation of cell lysis and measurement of luciferase activities were determined using the dual‐luciferase HS assay kit (GeneCopoeia). Luciferase activity was normalized with RLuc activity within each sample. In the separate experiments, SP1 promoter plasmids (0.6 μg/ml each) with Renilla luciferase reporters as an internal control were purchased from GeneCopoeia (Rockville, MD, USA). MO2‐CCAT1 (0.8 μg/ml), the control vector, and miR7‐5p mimics or negative controls, purchased from GeneCopoeia were cotransfected into the cells with the lipofectamine 3000. A luciferase activity assay was performed using the Luc‐Pair™ dual‐luciferase HS assay kit (GeneCopoeia) according to instructions from the manufacturer. Firefly luciferase activity was normalized to Renilla luciferase activity among every sample. Each experiment was repeated at least three times in triplicate.