Pmir reporter control vector

The 3′-UTR of the human MICA cDNA containing the putative target site for GATA-2 and GATA-3 was inserted into the pMIR-Reporter-control vector (Promega, Madison, WI, USA) immediately downstream of the luciferase gene. HepG2 or HepG2.2.15 cells were transfected with PGL3-basic, PGL3-MICA promoter or PGL3-MICB promoter together with pRL-TK using Lipofectamine 2000 according to the manufacturer's protocol. Luciferase activity was measured at 36 h after transfection using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Three independent experiments were performed in triplicate.

The 3´-UTR of human PD-L1 cDNA containing the putative target site for the miR-200c was inserted into the pMIR-Reporter-control vector (Promega, Madison, WI, USA) immediately downstream of the luciferase gene. HBV⁺HLCZ01 cells were transfected with reporter plasmids containing the 3´-UTR of PD-L1 (pMIR-Reporter-PD-L1-3´-UTR) and pRL-TK (Promega), together with 100 nM of miR-200c mimics, miR-200c inhibitors, or NCs utilizing Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, respectively. For the STAT3 or SALL4 target efficiency assay, the HLCZ01 cells were transiently co-transfected with a 0.1 μg pmiR-200c-Reporter working vector containing the wild-type sequence or site mutant sequence of the target gene. Luciferase activity was measured at 36 h after transfection utilizing the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well.