Luciferase reporter gene assay system

After amplification, circ-FKBP5 and RUNX2 sequences were cloned into pmirGLO plasmids (Promega) to gain circ-FKBp5-wild-type (WT) and RUNX2-WT. A fragment covering the mutant target region was designed and cloned into pmirGLO to gain circ-FKBP5-mutant-type (MUT) and RUNX2-MUT. Subsequently, co-transfection of miR-205-5p mimic or mimic NC with circ-FKBP5-MUT or RUNX2-WT or their respective MUT plasmids was done into BMSCs using Lipofectamine 2000. The relative luciferase activity was analyzed after transfection of 48 h in the luciferase reporter gene assay system (Promega) [26 (link)].

The luciferase activity assay verified the binding of miR-30b-5p to TGFBR2. The binding sites predicted by the software were inserted into the PMIR-Report luciferase REPORT vector, which were named WT-TGFBR2 and MUT-TGFBR2, respectively. Then the luciferase gene assay was used to co-transfect the cells with a 200 ng luciferase reporter gene vector and 25 ng PRL-TK (expressing luciferase as an internal control) using Lipofectamine 2000. Twenty-four h after luciferase transfection, the luciferase reporter gene assay system (Promega) was used to analyse enzyme activity.

A luciferase reporter for NFκB activity (pNFκB-luc) was a kind gift from Prof. Jiahuai Han, Xiamen University whereas reporters for interferon activity (pISRE-luc, pGL3-IFNα-luc and pGL3-IFNβ-luc) were provided by Prof. Rongtuan Lin, McGill University, Montreal, Canada. Luciferase reporter activities in cells co-transfected with any of the luciferase reporter were determined using a luciferase reporter gene assay system (Cat#E1601, Promega, Madison, WI, USA) as instructed. For all luciferase assays, β-galactosidase activities were determined to calibrate for the transfection efficiency. The calibrated value for a proper control was used to normalize all other values to obtain the normalized relative luciferase units (RLU).

NLRP3 promoter activity was analyzed by luciferase assay as previously described^{48 (link)}. Briefly, human NLRP3 gene promoter were inserted into the firefly luciferase vector pGL3-basic (Promega), termed pGL3-NLRP3. The human leukemia cell line (HL-60) is used as an alternative to primary neutrophils as described^{49 (link)}. Briefly, HL-60 was differentiated into neutrophil-like cells in the medium of RPMI) 1640 plus l-glutamine and 25 mM HEPES (Fisher Scientific) supplemented with antibiotic/antimycotic (Invitrogen) and 15% heat-inactivated fetal bovine serum (FBS) (Invitrogen), plus Dimethyl sulphoxide (DMSO), endotoxin, and hybridoma tested (Sigma). Neutrophil-like cells were then transfected by pGL3-NLRP3 plasmid using Lipofectamine 2000 Reagent (Invitrogen). After 48 h, the cells were detected by a luciferase reporter gene assay system (Promega). Normalization of firefly luciferase activity was based on Renilla luciferase activity.

The 3'-UTR sequence of NOTCH3 containing the miR-7-5p binding site was cloned and constructed into a pGL3 vector (E1910, Promega, USA) to generate a wild-type NOTCH3 reporter gene (NOTCH3-Wt). GeneArt™ site-directed mutagenesis system (Thermo Fisher Scientific) was used to generate the mutant NOTCH3 reporter gene (NOTCH3-Mut). NOTCH3-Wt or NOTCH3-Mut and miR-NC and miR-7-5p mimic enter EC cells [15 (link)]. 48 hours after transfection, 40 μL stop reagent Renilla luciferase was added after the determination of the firefly luciferase fluorescence value, and the luciferase activity was measured by the luciferase reporter gene assay system (Promega, Madison WI, USA).