Pgl3 basic luciferase vector

The ANXA2 3′-untranslated region (UTR) and the full miR155HG sequence containing miR-185-5p seed matching sites were amplified from human cDNA via PCR and cloned into the 3′ end of the pGL3-basic luciferase vector (Genechem). Mutated versions of each construct were generated by mutating the miR-185-5p seed site sequences (pGL3-wt or mut). The miR155HG promoter region sequence (2000 bp to 1000 bp upstream of transcription starting point) were also amplified and cloned into the 5′ end of the pGL3-basic luciferase vector. Mutated version was generated by deleting binding region sequences of p-STAT3 (wt or mut-pGL3). U87 cells seeded into 96-well plates were co-transfected with wt or mut report gene, the pRL-TK control (Promega, Madison, WI, USA) and miR-185-5p mimic or miRNA NC using Lipofectamine 2000 (Invitrogen). The wt or mut-pGL3 and the pRL-TK control were co-transfected into the cells, then treated with cell culture with or without SH-4-54 inhibitor of STAT3 phosphorylation. At 48 h after transfection, luciferase activity was determined using the Dual Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer’s protocol. The relative luciferase activity was normalized to Renilla luciferase activity. All assays were performed in triplicate.

The LINC00689 promoter sequence containing KLF15 seed matching sites (LINC00689-WT) was amplified via PCR and cloned into the pGL3-basic luciferase vector (GeneChem, Shanghai, China). The mutant reporter plasmid was also generated by mutating KLF15 seed site sequences (LINC00689-MUT). HCT116 and LoVo cells seeded in 6-well plates and grown to around 80% confluence, then cells were co-transfected with OE-KLF15 or OE-NC plasmids and recombinant reporter plasmids (LINC00689-WT/MUT) for 48 h using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Afterwards, cells were harvested and luciferase activities were measured using a Luciferase Reporter Assay Kit (Promega, Madison, WI, USA). Briefly, the transfected HCT116 and LoVo cells were lysed and centrifuged to remove cell debris. The supernatant was transferred to a luminometer plate and luciferase substrate from the Luciferase Reporter Assay Kit was added. Luciferase activity was measured using a luminometer (Thermo Fisher Scientific).