Dual luciferase reporting system

The wild‐type (wt) or mutated (mut) mir‐21‐5p binding sequences from SMAD7 3′UTR were cloned into the pGL3 basic vector. Cells seeded at a density of 50% were placed into a 12‐well culture plate. After 16 h of continuous culture, the luciferase reporter gene plasmid and pRL‐TK plasmid were transfected when the cell density was about 70%. According to the manufacturer's protocol, Lipofectamine 2000 was used to cotransfect the cells with mir‐21‐5p mimics, SMAD7 (wt) and SMAD7 (mut). The renilla luciferase was used as a loading control. The cells were harvested. Luciferase activity was analyzed using a dual luciferase reporting system (Promega). In brief, after cotransfection of the plasmid for 48 h, the medium was discarded and the cells were washed with PBS. The 12‐well plate was tilted and the remaining PBS aspirated. Then, 50 μl PLB was added to each well and shaken for 30 min on a shaker to ensure complete cell lysis. In a white opaque 96‐well plate, 10 μl supernatant and 100 μl luciferase assay reagent II was added to each well, and the luciferase intensity measured. After the test had been completed, we added 100 μl Stop&Glo reagent to each well, and measured the renilla luciferase activity.

The online program Target-scan 7.0 predicted that there was a binding site between miR-206 and HDAC4 3′UTR, so HDAC4 was speculated to be the potential target gene of miR-206. Then, we selected the luciferase reporter gene assay to verify this conjecture. The 3′-UTR of HDAC4 was cloned into PGL3 luciferase reporter gene vector to build wild type (WT) and mutant type (MT) of HDAC4 3′-UTR luciferase report vectors. Then the cells were co-transfected with the WT-HDAC4 or MUT-HDAC4 and miR-206 inhibitor or miR-NC. After incubation for 48 h, the luciferase activity of each group was detected in strict accordance with the instructions of the dual-luciferase reporting system (Promega, INC., USA). The fluorescent intensity of renal cells was used as an internal reference.

The binding sequence between miR-4429 and DLX1 mRNA was first predicted on a bioinformatic system Starbase (http://starbase.sysu.edu.cn/). To validate the binding relationship between miR-4429 and DLX1 mRNA, DLX1 3′UTR containing the putative binding site with miR-4429 was designed and inserted into pmirGLO reporter vectors (LMAIBio Co., Ltd., Shanghai, China) to construct wild-type luciferase vectors. A mutant type vector was designed by mutating the binding sequence between miR-4429 and DLX1 mRNA. Well-constructed reporter vectors were co-transfected with miR-4429 mimic or mimic control into PCa cells. Forty-eight hours later, the cells were harvested and lysed, and luciferase activity in cells was determined on a dual luciferase reporting system (Promega Corp., Madison, Wisconsin, USA) according to the manufacturer's protocol.

The dual-luciferase assay was used to confirm whether NONHSAT030515 directly targeted miR-490-5p and whether miR-490-5p directly targeted BMPR2. HADSCs were co-transfected with NONHSAT030515 (BMPR2) wild or mutant plasmids, miR-490-5p control, or miR-490-5p mimics. Luciferase activity was assessed 48 h after transfection using a dual-luciferase reporting system (Promega Company).

The binding sites of USF1 on the HAS2-AS1 promoter region were cloned into restriction enzymes Kpnl and HindIII digestion sites 200 on firefly luciferase reporter vector pGL3 (Promega, Madison, WI, U.S.A.). According to the manufacturer’s instructions, the above vectors and the USF1 expression vector (USF1 construct) or the negative control (empty vector) were co-transfected into U87 and U251 cells instantaneously by Lipofectamine 2000 (Invitrogen). The relative luciferase activity was evaluated using a dual luciferase reporting system (Promega, Madison, WI, U.S.A.) 48 h after transfection.