Pmirglo reporter plasmid

Neurod4 was predicted as a target gene of miR-340-5-5p using TargetScan software (version 7.2; http://www.targetscan.org/vert_72/). Thus, a dual-luciferase reporter assay was performed to verify the target binding of miRNA-340-5p and Neurod4. The wild-type (WT) 3′ untranslated region (3′UTR) binding site of Neurod4 was amplified using PCR and cloned into a pmirGLO reporter plasmid (Promega Corporation). The 3′UTR fragment of Neurod4 was also mutated, resulting in a mutant (MUT) 3′UTR, using the Fast MultiSite Mutagenesis System (Beijing Transgen Biotech Co., Ltd.) and cloned into the pmirGLO reporter plasmid. Cells (5×10⁴) cultured in 24-well plates were co-transfected with an equal concentration (450 ng/µl) of Neurod4 (WT or MUT) and miR-340-5p mimic or miR-NC using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. After transfection for 48 h, the relative luciferase activity was detected using a Dual-Luciferase Reporter assay system (Promega Corporation), according to the manufacturer's protocols. Luciferase activity was normalized to Renilla luciferase activity.

The MicroRNA.org database (http://www.microrna.org/) was used to analyze and predict the target gene of miR-183. Target sequences of OSMR-wild-type (WT) 3′untranslated region (UTR) and OSMR-mutant (MUT) 3′UTR were constructed manually and inserted into the pmirGLO reporter plasmid (Promega Corporation, Madison, WI, USA) by double enzyme digestion of the restriction site BamHI/HindIII to obtain the WT and MUT plasmids. The two plasmids were co-transfected with miR-183 mimic and mimic control into 293T cells (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., Shanghai, China), using Entraster™ reagent (Engreen Biosystem Co., Ltd., Beijing, China). At 48 h post transfection, the cells were collected and lysed, and luciferase activity was detected using a Dual-Luciferase Reporter Assay system (cat. no. E1910; Promega Corporation). To the cells, 100 µl firefly luciferase solution was added to detect the activity of firefly luciferase and 100 µl Renilla luciferase solution was added to detect the activity of Renilla luciferase. The relative luciferase activity was calculated. The experiment was repeated three times.