Glomax bioluminescence detector

HEK293T cells (Procell, Wuhan, China) were used for the dual luciferase assay. Wild-type and mutant plasmids were constructed and named psiCHECK2.0-NONMMUT100923.1, psiCHECK2.0-mut-NONMMUT100923.1, psiCHECK2.0-Cdsn, and psiCHECK2.0-mut-Cdsn (n = 3) (Sangon Bioengineering Co., Ltd., Shanghai, China). Each plasmid was co-transfected into HEK293T cells together with miR-200a-3p mimic, blank, and mimic NC. Luciferase activity was detected using a Dual-Luciferase Reporter Assay System (116680119; Invitrogen). Firefly luciferase (F) and Renilla luciferase (R) activities were detected using a manual dual fluorescence detector (GloMax bioluminescence detector, Promega), and the activity multiple was calculated.

The mRNA 3′UTR of FUT2 was amplified and sub-cloned into the pMIR-GLO reporter plasmid (Promega Corporation), and the resulting plasmids were named FUT2-WT and FUT2-MUT. Chondrocytes were cultured in a 96-well plate and co-transfected with miR-4428 mimics/NC and FUT2-WT/FUT2-MUT using Lipofectamine 2000 (Life Technologies; Thermo Fisher Scientific, Inc.). After 48 h, luciferase activity was detected using a dual-luciferase reporter assay system (Promega Corporation). The 20 µl cell lysate was added to the luminescent plate. Background values were read with a GloMax bioluminescence detector (Promega Corporation) and 100 µl LARII solution (Promega Corporation) were added to each sample. After reading, add 100 µl Stop&GloR Reagent (Promega Corporation) was added to each sample. Normalization was carried out according to Δ activity multiple=(F/R) sample (/F/R) for comparison (F, Firefly luciferase; R, Renilla luciferase).

Dual-Luciferase reporter assay was performed to validate the binding of miRNA 18a-5p with the 3′-UTR region of EPHA7 in WM266 and A375 cells. The wild-type (WT) and mutated (MUT) 3′-UTR regions of EPHA7 were ligated separately with the Psi-CHECK2 plasmids (Promega Corporation), which were then transfected into WM266 and A375 cells (1×10⁵ cells/well) using Lipofectamine 2000 as aforementioned, together with miRNA 18a-5p mimics or NC as designated. The cells were cultured under normal conditions at 37°C for 48 h, followed by rinsing three times with PBS and incubation with passive lysis buffer. A GloMax^® Bioluminescence detector (Promega Corporation) was used to measure the luciferase activity of cell lysates. The luciferase activity was normalized to Renilla luciferase activity.