Dual luciferase reporter system

After hBMSCs were cultured for 24h, the wild type (wt) and mutant type (mut) NEAT1 sequence were cloned into pmirGLO reporters (GenePharma, China) containing the predicted binding sites of miR-339-5p and SPI1 using a Lipofectamine 2000 kit (GenePharma, China) with renilla luciferase served as an internal normalizer. 48 h post transfection, a dual‑luciferase reporter system (GenePharma, China) was used to examine luciferase activities.

MG63 and U2OS cells were seeded into 24-well plates. After 24 h of incubation, 6 ng pmirGLO report vector (Shanghai GenePharma Co., Ltd.) carrying the wild-type (wt) or mutant (mut) 3′-untranslated region (3′-UTR) of TPX2 was cotransfected with miR-29c-3p mimics (100 nmol/l) or miR-29c-3p inhibitors (100 nmol/l) into the osteosarcoma cells using a Lipofectamine 2000 kit (Invitrogen; Thermo Fisher Scientific, Inc.). The negative controls for the miRNA mimics and inhibitors were non-targeting sequences. The sequences of the mimics, inhibitors and negative controls were as follows: miR-29c-3p mimic sense, 5′-UAGCACCAUUUGAAAUCGGUUA-3′ and antisense, 5′-UAACCGAUUUCAAAUGGUGCUA-3′; negative control mimic sense, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′ and anti-sense, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′; miR-29c-3p inhibitor, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′; and negative control inhibitor, 5′-UAACCGAUUUCAAAUGGUGCUA-3′ (Shanghai GenePharma Co., Ltd.). The amplified TPX2 wt or mut 3′UTR containing the miR-29c-3p target sites was inserted into the pMIR-GLO luciferase reporter vectors (Shanghai GenePharma Co., Ltd.). At 48 h post-transfection, luciferase activities were examined with a dual-luciferase reporter system (Shanghai GenePharma Co., Ltd.) and normalized with Renilla luciferase activity using SpectraMax i3 (Molecular Devices LLC).