Site directed mutagenesis kit

Dual-Luciferase reporter assays (Promega, Madison, WI, USA) were used following the manufacturer’s instructions. The putative miR-19b-3p complementary site in the 3′-UTR of SMAD4 mRNA (NM_005395; 3′-UTR: 1352–1358) or its mutant sequence were cloned into the pmirGLO luciferase reporter vector. The novel combined plasmid was named pmirGLO-SMAD4 3′’-UTR-wt (wild type). A mutation of the 3′-UTR of SMAD4 was created by using a site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and designated as pmirGLO-SMAD4 3′-UTR-mut (mutant). SW480 cells were plated at 4000 cells per well in 100 μL DMEM in a 96-well microplate (BD Biosciences, USA). Twenty-four hours after plating, cells were co-transfected with 0.5 μg of pmirGLO-SMAD4 3′-UTR-wt or pmirGLO-SMAD4 3′-UTR-mut, 0.01 μg of the pMirGLO-Vector (Promega, Madison, WI, USA), 50 nM miR-19b-3p mimics (Mimics), and miRNA mimics Negative Control (NC) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The firefly and Renilla luciferase activities were measured 24 h after transfection.

The 5′UTR region of TGFB1 was cloned into the pGL3–basic vector in the front of the F-Luc gene (pGL3–basic–WT–5′UTR). The CDS region of TGFB1 was cloned into the pcDNA3–HisMax vector (pcDNA3–WT–CDS). Mutagenesis of m⁶A motifs (GGAC to GGCC) in both pGL3–basic–WT–5′UTR and pcDNA3–WT–CDS was performed using a site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, US). Primers for mutagenesis are listed in Additional file 1: Table S1. The CDS region of the TGFβ binding protein (LTBP1) was cloned into the pHM6 vector.

RIE-1, HeLa, and NMuMG cells were infected with a V5-TrkB using a pLenti6.3/V5-TOPO TA Cloning Kit (Invitrogen, Waltham, MA, USA) to generate TrkB overexpression cells [25 (link)] and the TrkB K588M mutant using previously described [40 (link)] was generated by site-directed mutagenesis with a Site-Directed Mutagenesis Kit (ThermoFisher Scientific, Lafayette, CO, USA). To generate a stable knockdown of TrkB, small hairpin-expressing vectors were purchased from Sigma-Aldrich (SHCLNG-NM_006180). Production and infection of target cells were previously described [25 (link)] and selected with 2 µg/mL puromycin and 500 µg/mL G418. Plasmid transfections were carried out using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) reagent according to the manufacturer’s instructions.

To investigate MALAT1 and miR-206 interaction, MALAT1 sequence containing miR-206 binding sites was inserted into a pmirGLO dual luciferase vector (Promega, Madison, WI, USA) to generate wildtype (WT) pmirGLO-MALAT1. The mutant (MUT) of MALAT1 sequence in miR-206 binding sites was synthesized using Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, Norristown, PA, USA) and inserted into a vector to generate MUT pmirGLO-MALAT1. Custom miR-206 mimics and inhibitors were purchased from Horizon Discovery (Boyertown, PA, USA). The pmirGLO vectors containing WT- or MUT-MALAT1 sequence were co-transfected with miR-206 mimic and miR-206 inhibitor to cancer cells by Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). The relative luciferase activities were measured by Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI, USA). To investigate miR-206 and MCP-1, the 3′UTRs of MCP-1 containing the predicted miR-206-binding sites and MUT sites were, respectively, inserted into pmirGLO dual luciferase vector pmirGLO-MCP-1-3′UTR-WT and pmirGLO-MCP-1-3′UTR-MUT. The 3′UTRs of MCP-1 with predicted miR-206-binding sites and their mutant versions were cloned into pmirGLO vectors. These constructs were co-transfected into lung cancer cells with miR-206 mimic and inhibitor. The same Dual-Luciferase Reporter Assay was used to evaluate the regulatory effect of miR-206 on MCP-1.