Pmirglo dual luciferase mirna target expression vector system

Luciferase constructs were made using the pmirGLO Dual-Luciferase miRNA Target Expression Vector System (Promega, Madison, USA). A YOD1 3′ UTR fragment was generated by RT-PCR from MV-4-11 cell total RNA. The primer sequences are provided in Supplementary Table 5. Luminescence was measured using a dual-luciferase reporter assay system (Promega).

To validate the direct binding of let‐7d with TGFβRI gene promoter region, the dual‐luciferase reporter assay was carried out using the pmirGLO Dual‐Luciferase miRNA Target Expression Vector System (Promega). The recombinant pmirGLO‐WT (wild‐type) TGFβRI and pmirGLO‐Mut (mutant) TGFβRI plasmids were established separately following the manufacturer's instructions, which were then transfected into the mouse lung microvessel pericytes using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) as instructed by the manufacturer. Subsequently, cells transfected with different recombinant plasmids were transfected with the let‐7d mimic using the Lipofectamine 3000 reagent as well, followed by the detection of the luciferase activities using a GloMax^® 20/20 Luminometer (Promega).