Luciferase reporter plasmids, transfection, and luciferase assay

The 3′ untranslated region (3′UTR) of human TGF‐β, TGFBR2, IL‐1β, and ET‐1 gene containing the putative miR‐483 targeting sites was synthesized by Shanghai Personal Biotechnology and subcloned into the pmirGLO dual‐luciferase miRNA target expression vector (Promega) to obtain Luc‐TGF‐β‐3′UTR‐WT, Luc‐TGFBR2‐3′UTR‐WT, Luc‐IL‐1β‐3′UTR‐WT, and Luc‐ET‐1‐3′UTR‐WT reporter constructs. Mutant constructs were further created with “GTGATCGC” to “GGGGTGGC” replacement in Luc‐TGF‐β‐3′UTR‐mut; “AGAGGAGTG” to “TAGTACTAT” in Luc‐TGFBR2‐3′UTR‐mut; “CTGTTGTCT” to “CAGTAGACA” in Luc‐IL‐1β‐3′UTR‐mut; and “GGAGTG” to “GCAATC” in Luc‐ET‐1‐3′UTR‐mut. The various reporter constructs were transfected into BAECs by using Lipofectamine 2000 (Invitrogen). The luciferase activity was measured by using the Dual‐Glo Luciferase Reporter Assay Kit (Promega) with a luciferase reader (PerkinElmer).