Mir 21 nc

We seeded 293T cells in 24-well plates, cultured then overnight, and transfected them with the wildtype and mutant TRPM7 promoter-luciferase plasmids (0.1 μg per well of pGV272-wt-TRPM7 or pGV272-mt-TRPM7 plasmids) mediated by Lipofectamine 2000 (Invitrogen, CA, United States). Meanwhile, cells were cotransfected with either 0.4 μg miR-21 mimics or 0.4 μg miR-21 NC (Genepharma, Shanghai, China). Transfection efficiency was standardized by cotransfection with 0.02 μg Renilla luciferase reporter (Genechem, Shanghai, China). Both firefly and Renilla luciferase activities were quantified using a dual-luciferase assay system (Promega, E1910).

Human normal lung epithelial cells BEAS-2B and human LUAD cells A549 and H1299 were purchased from the National Collection of Authenticated Cell Cultures. BEAS-2B, A549, and H1299 cells were cultured in DMEM (Hyclone, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 100 mg/mL penicillin, and 100 mg/mL streptomycin. The medium was placed in an incubator with 5% CO₂ at 37°C.
A549 and H1299 cells were treated with 25 nM, 50 nM, and 100 nM calycosin [15 (link)] for 24 h when the confluence reached 65%-80%. Then, subsequent functional assays were conducted. The circ_0001946 interfering fragment (si-circ_0001946) and its control (si-NC), miR-21 mimics and its control (miR-21 NC), and miR-21 inhibitor and its control inhibitor were designed and synthesized by GenePharma (Shanghai, China). Cells were seeded and cultured in 6-well plates until their confluence reached 65%-80%. Subsequently, the above fragments or vectors were transfected into A549 and H1299 cells using Lipofectamine 2000 (Invitrogen, USA). After 48 h culture, the cells were collected. In addition, to verify the action mechanism of calycosin, A549 cells were first treated with 100 nM calycosin for 24 h and then transfected with miR-21 inhibitor and si-circ0001946.