Mir 499a 5p inhibitor

The binding sites of miR-499a-5p and TMEM189 were predicted by TargetScan software. TMEM189 WT, TMEM189 MUT, miR-499a-5p mimic, miR-499a-5p inhibitor and control vectors (GenePharma) were designed and constructed. Next, pmirGLO-TMEM189-WT and pmirGLO-TMEM189-Mut plasmids were co-transfected into MDA-MB-231 with miR-499a-5p mimic and mimic-NC. Cell lysates were collected 48 h after transfection. Luciferase activity was assessed using a dual-luciferase reporter gene assay system (Promega, Madison, WI). In this experiment, renilla luciferase activity was selected as the internal reference.

pcDNA3.1/LINC01018, a miR-499a-5p mimic (5′-UUAAGACUUGCAGUGAUGUUU-3′), miR-499a-5p inhibitor (5′-AAACATCTCTGCAAGTCTTAA-3′), miR mimic control (5′-ACUACUGAGUGACAGUAGA-3′), miR inhibitor control (5′-CAGUACUUUUGUGUAGUACAA-3′), si-PDCD4 (sense, 5′-GGAGCGGUUUGUAGAAGAAdTdT-3′ and antisense, 3′-dTdTCCUCGCCAAACAUCUUCUU-5′), si-control (sense, 5′-UUCCCUUUGUCAUCCUUUGCCUdTdT-3′ and antisense, 3′-dTdTAAGGGAAACAGUAGGAAACGGA-5′) and a pcDNA3.1 empty vector were all purchased from Shanghai GenePharma Co., Ltd. HL-60 and THP-1 cells were transfected with pcDNA3.1 (50 ng), pcDNA3.1/LINC01018 (50 ng), miR-499a-5p mimic (100 nM), mimic control (100 nM), miR-499a-5p inhibitor (100 nM), inhibitor control (100 nM), si-PDCD4 (50 nM), and si-control (50 nM) at 37°C for 48 h using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected and used 48 h later for subsequent experimentation.