Renilla luciferase expression of prl tk plasmids

The luciferase reporter plasmids [wild-type (wt)-PTEN-UTR-pGL3 or mutant (mut)-PTEN-UTR-pGL3] were synthesized by Shanghai GenePharma Co., Ltd. 293T cells (8×10⁴) (American Type Culture Collection) were co-transfected with the luciferase reporter along with miR-93 mimics/inhibitor using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, the activity of luciferase was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase expression of pRL-TK plasmids (Promega Corporation) was used for normalization.

The miR-106a-5p mimics/inhibitor and the corresponding negative controls (NCs) were designed and synthesized by Shanghai GenePharma Co., Ltd. The fragment of the 3′-UTR of STAT3 [wild-type (wt) or mutant (mut)] was amplified and cloned into the pMIR-REPORT luciferase vector (Ambion; Thermo Fisher Scientific, Inc.). Site-directed mutagenesis of the STAT3 3′-UTR at the putative miR-106a-5p binding site was performed by a QuikChange kit (Qiagen, Inc.). Subsequently, HUVECs at a density of 2×10⁵/well were seeded into 24-well plates and co-transfected with 0.8 μg of pMIR-STAT3-wt-3′-UTR or pMIR-STAT3-mut-3′-UTR, 50 nM miR-106a-5p mimic/inhibitor or the corresponding NC using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The relative firefly luciferase activity was measured 48 h after transfection by using the Dual-Light^® luminescent reporter gene assay (Applied Biosystems; Thermo Fisher Scientific, Inc.). Renilla luciferase expression of pRL-TK plasmids (Promega Corporation) was used for normalization.