β galactosidase expression plasmid

The entire 3′-UTR of human KRAS was amplified via PCR with human genomic DNA as a template. Then, the PCR products were inserted into the pMIR-REPORT plasmid (Ambion, USA), and successful insertion was confirmed by DNA sequencing. For the assessment of binding specificity, the KRAS sequence known to bind to the seed region of miR-548d-3p was mutated (from GCAAAAGTA to CGTTTTCAT), and the mutant KRAS 3′-UTR was inserted into an equivalent luciferase reporter.
For the luciferase reporter assays, hFOB 1.19 cells were seeded into 24-well plates. The cells were co-transfected with 1 μg of the firefly luciferase reporter plasmid, 1 μg of the β-galactosidase expression plasmid (Ambion) and equal amounts (100 pmol) of miR-548d-3p mimics, inhibitors or control miRNA by means of Lipofectamine 2000 (Invitrogen). The β-galactosidase plasmid was used as a transfection efficiency control. After the cells were incubated for 24 h, luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega, Beijing, China). The experiments were repeated independently three times.

Luciferase vectors were purchased from Genescript (Nanjing, China). Briefly, for miRNA binding site assays, luciferase reporter gene plasmids harboring the wild-type 3'UTR of ATG10, ATG4C, ATG2A or ATG2B were constructed. We also constructed a mutant 3'UTR of ATG10, which was mutated from ACUGUGA to TGACACT. For the miR-27B promoter activity assay, miR-27B promoter regions containing different c-Myc binding sites were inserted into pGL3-Basic reporter gene vectors from Genescript (Nanjing, China). We cotransfected SW480-OxR cells with Luciferase vectors, small RNA oligos and a β-galactosidase expression plasmid (Ambion, Carlsbad, CA, USA). Twenty-four hours after transfection, Luciferase activity was measured using a luciferase assay kit (Promega, USA).

The human FasL luciferase reporter construct (HFLP-Luc) containing 1.2 kb of the FASLG promoter, and the empty control plasmid HsLuc have been described previously.^{38 (link)} HFLP-Luc with single or double mutated LRH-1 binding sites in the FASLG promoter (HFLP mut BS1, HFLP mut BS2, HFLP mut BS1+2) were generated by site-directed mutagenesis using a kit from Stratagene (Quick-Change, Agilent) with primers shown in Table 1. The putative LRH-1 binding sites and in the FASLG promoter and their mutation are depicted in Figure 2a.
The LRH-1 reporter containing 5 copies of the LRH-1 binding motive of the human SHP promoter in the pGL3 basic plasmid (Promega, Mannheim, Germany) has been described previously.^{39 (link)} The Myc/6xHis-tagged LRH-1 expression plasmid was generated by cloning human LRH-1 into a pcDNA3.1 Myc/His expression vector (Invitrogen). The FLAG-tagged LRH-1 expression plasmid was generated by exchanging the Myc/His tag with a 3xFLAG epitope tag. A β-galactosidase expression plasmid was used to normalize transfection efficiency (Invitrogen).