Pmirglo luciferase reporter vector

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).

Luciferase Assay System (GenePharma Co., Ltd., Shanghai, China) was used to confirm the target relationship between miR-29b and the potential downstream target genes. Briefly, wild type 3′-UTR sequence of target genes containing miR-29b binding site and mutant 3′-UTR sequence of those target genes were inserted into Pmir-GLO luciferase reporter vectors (GenePharma Co., Ltd., Shanghai, China). These Pmir-GLO vectors were co-transfected with miR-29b mimic/inhibitor or mimic/inhibitor control (GenePharma Co., Ltd., Shanghai, China) into human bronchial epithelial cells (16HBE cells). The cells were harvested and relative luciferase activity (activity firefly/activity renilla) was detected.

The fragment of the 3ʹ-UTR of human CD55 containing the predicted target site of miR-132-3p (wild-type coding region 3ʹ-UTR) or CD55 subjected to site-directed mutagenesis of the 3ʹ-UTR (mutant coding region 3ʹ-UTR) was amplified by RT-qPCR. The PCR product was cloned downstream of the modified pmirGlO luciferase reporter vector (GenePharma, Shanghai, China). All constructs were confirmed by DNA sequencing.
For the luciferase assay, 293T cells were seeded into 24-well plates at a density of 1 × 10⁵ cells/well. At 24 h after plating, cells were co-transfected with 50 nM miR-132 mimics or miR-NC mimics and 500 ng of CD55 3ʹ-UTR wild-type or CD55 3ʹ-UTR mutant-type plasmids per well using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 24 h after transfection using the Dual-luciferase Reporter Assay System (Promega, Fitchburg, WI, USA), according to the manufacturer’s instructions. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity to control for the transfection efficiency.

Online bioinformatics software TargetScan (version 3.1; www.targetscan.org/mamm_31) was used to predict that miR-375 contained the binding sites in the 3′UTR region of RAC1.
The RAC1 3′-UTR-wild-type (WT) and RAC1 3′UTR-mutant (Mut) sequences were inserted into the pmirGLO luciferase reporter vector (Shanghai GenePharma Co., Ltd.). Subsequently, HSCs (1×10⁶) were co-transfected with 1 µg miR-375 mimic, pre-NC, miR-375 inhibitor or NC and 1 µg RAC1 3′UTR-WT or RAC1 3′UTR-Mut using Lipofectamine 2000 transfection reagent. At 48 h post-transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.