Pgl3 reporter luciferase vector

TargetScan 7.2 (http://www.targetscan.org) used to predict potential binding targets of miR-29b. The wild-type (WT) or mutant (mut) 3'-UTR of TGF-β1 was cloned into the pGL3 luciferase reporter vector (Shanghai GeneChem Co., Ltd.). The corresponding mutant sequence was designed to be identical to that of miR-29b to prevent sequence-specific binding. The pGL3-TGF-WT or pGL3-TGF-mut plasmid (1 µg cDNA) was co-transfected with miR-29b mimic (20 pmol RNA) into cultured VSMCs A firefly-Renilla dual luciferase assay was performed on samples 48 h after transfection to determine luciferase activities, using a Dual-Luciferase^® Reporter assay system (Promega Corporation) following the manufacturer's protocol. Results were detected using a Synergy 2™ Microplate Reader (BioTek Instruments, Inc.).

The binding sites between lncRNA XIST and miR-92a, as well as miR-9a and Itgα5 or KLF4, were predicted using DIANA-LncBase v.2. A WT and Mut lncRNA XIST sequence and a WT and Mut 3′ UTR fragment of Itgα5 or KLF4 containing the putative miR-92a binding site were synthesized by Genechem (Shanghai, China). The constructs were cloned into the pGL3 luciferase reporter vector (Genechem, Shanghai, China) to generate XIST-WT, XIST-Mut, Itgα5-WT, Itgα5-Mut, KLF4-WT, and KLF4-Mut luciferase reporter systems. The mutations were confirmed by sequencing. Then, the luciferase reporter plasmids were co-transfected with miR-92a-M or NC-M (GenePharma, Shanghai, China) into the HEK293T cells. After 48 h of transfection, the luciferase activity was determined using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The relative luciferase activity was normalized to Renilla luciferase internal control.