Dual luciferase reporter assay system

The wild type and mutant regions of OGT promoter were amplified by PCR and cloned into pGL3 basic vector (#E1751, Promega; Madison, WI, USA). HEK-293 T cells were co-transfected with Renilla luciferase expression plasmid (pRL-TK, #D2760, Beyotime) and TWIST1 overexpression vector. After 48 h, luciferase activity was measured with a Dual Luciferase Reporter Assay System (#RG028, Beyotime).

Human embryonic kidney 293T cells (HEK293T) were cultured in the following conditions: Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) grown in a humidified atmosphere of 5% CO₂ at 37°C.
The wild-type 3′UTR fragments of 12 candidate target genes containing predicted cas-miR4018 or cas-miR4000f seed sites were amplified by PCR using cDNA as templates. The PCR products were cloned into the firefly luciferase coding region of the pmirGLO plasmid to construct reporters for luciferase assays. For the luciferase assay, 6 h before cell transfections, HEK293T cells were passaged in 96-well plates with 3 × 10⁴ cells per well. Then the cells were cotransfected with 0.1 μg of luciferase reporter plasmid and 100 nM miRNA mimics. Twelve hours after transfection, a fresh medium was placed into each well. Forty-eight hours after transfection, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Beyotime, China), and relative reporter activity was normalized to Renilla luciferase activity. The data statistical analyses were performed using paired Student’s t-tests. Experiments were performed in triplicate.

Normal and mutant 3′UTRs (untranslated regions) of the PSMB8 gene (Gene ID: 102180902) in goats were amplified by PCR and inserted downstream of the luciferase gene in the pGL3 vector (Promega, Cat. No. E1751). The sequence of PSMB8 3′UTR and PSMB8-mut 3′UTR were as follows: 5′-CAATAAAGGAAAACGGTTA-3′ and 5′-CAATAAAGGAAGGTAACCA-3′. 293T cells used for the dual-luciferase reporter assay were purchased from CCTCC (Wuhan Province, China). Co-transfection with chi-miR-451-5p mimics (or chi-miR-451-5p mimics NC) and PSMB8-3′UTR (or PSMB8-Mut or pGL3 vector) was performed using Lipofectamine 3000 (Invitrogen, Cat. No. L3000015), together with 0.1 µg/well of pRL-TK (Beyotime, Cat. No. D2760) when the 293T cells reached a confluence of 75%. Forty-eight hours following the transfection, firefly luciferase activities and Renilla luciferase activities were measured continuously using a dual-luciferase reporter assay system (Beyotime, Cat. No. RG027) according to the manufacturer’s instructions on a modular multimode microplate reader (BioTek Synergy H1) at 560 nm and 465 nm, respectively. The firefly to Renilla luciferase ratio was used to measure relative activity.