Luc pair dual luciferase hs assay kit

The wild-type and mutation-type VEGFA 3′UTR luciferase vectors were synthesized (GeneCopoeia, MD, United States). SP1 promoter vector with Renilla luciferase reporter as internal reference was also generated by GeneCopoeia. Co-transfected these vectors into cells with either miR-125b-5p mimics or inhibitors and corresponding negative control using Liposomal Transfection Reagent (Yeasen, Shanghai, China). Detection of luciferase activities was undergone by using Luc-Pair™ Dual-Luciferase HS Assay Kit (GeneCopoeia MD, United States). Optical density was normalized with Renilla within each sample.

The wild and mutation types of SP1 3'‐UTR luciferase vectors (0.6 μg/ml each) obtained from GeneCopoeia, Inc. (Rockville, MD, USA) were transfected into the cells with either miR7‐5p mimics (100 nM) or negative control using lipofectamine 3000 reagent; the preparation of cell lysis and measurement of luciferase activities were determined using the dual‐luciferase HS assay kit (GeneCopoeia). Luciferase activity was normalized with RLuc activity within each sample. In the separate experiments, SP1 promoter plasmids (0.6 μg/ml each) with Renilla luciferase reporters as an internal control were purchased from GeneCopoeia (Rockville, MD, USA). MO2‐CCAT1 (0.8 μg/ml), the control vector, and miR7‐5p mimics or negative controls, purchased from GeneCopoeia were cotransfected into the cells with the lipofectamine 3000. A luciferase activity assay was performed using the Luc‐Pair™ dual‐luciferase HS assay kit (GeneCopoeia) according to instructions from the manufacturer. Firefly luciferase activity was normalized to Renilla luciferase activity among every sample. Each experiment was repeated at least three times in triplicate.

This procedure was reported previously (Tang et al., 2015). Briefly, NPC cells (2.5 × 10⁵ cells/well) were seeded in 6‐well dishes and grown to 60–70% confluence. The mimics of miR7‐5p (100 nM) and the negative controls purchased from Ribo Biological Co., Ltd. (Guangzhou, China), control and SP1 overexpression vectors (pCMV6‐SP1; 0.8 μg/ml), MO2‐CCAT1 (0.8 μg/ml) were transfected into the cells using lipofectamine 3000 transfection reagent (Invitrogen, Shanghai, China) for up to 30 hr based on the instruction from the provider, followed by treating with SM for an additional 24 or 48 hr for other experiments. In the separated experiments, control and wild type SP1 promoter constructs (purchased from GeneCopoeia, Inc., Rockville, MD, USA) with or without 0.2 μg of the internal control secreted alkaline phosphatase were cotransfected into the cells with the lipofectamine 3000 transfection reagent. The preparation of cell extracts and measurement of luciferase activities were determined using Luc‐Pair™ dual‐luciferase HS assay kit (GeneCopoeia, Inc., Rockville, MD, USA).