Mir 92a mimic

Bioinformatics software http://www.targetscan.org was used to predict the targeting relationship between miR-92a and TCF21 and the binding sites of miR-92a to TCF21 3′UTR. TCF21 3′UTR promoter sequence containing miR-92a binding site was synthesized and TCF21 3′UTR wild type plasmid (TCF21-WT) was constructed. On the basis of this plasmid, the mutant type plasmid TCF21 3′UTR (TCF21-MUT) was constructed. The procedure was carried out according to the instructions of plasmid extraction kit (Promega, Madison, Wisconsin, USA). The cells in the logarithmic growth were inoculated into the 96-well plate and transfected with Lipofectamine 2000 when the cell density was about 70%. TCF21-WT and TCF21-MUT were mixed with mimics NC and miR-92a mimics (Shanghai GenePharma Co., Ltd (Shanghai, China)) respectively, and then co-transfected into 293T cells. After 48 h of transfection, luciferase activity was collected and lysed. The luciferase activity was detected by a Glomax20/20 luminometer (Promega, Madison, Wisconsin, USA) with a luciferase detection kit (BioVision, San Francisco, CA, USA). The experiment was repeated three times.

TUG1 lentivirus overexpression vector (LV‐TUG1), TUG1 knock‐down vector (sh‐TUG1), miR‐92a mimic (50 nM), miR‐92a inhibitor (50 nM), FXR1 lentivirus overexpression vector (LV‐FXR1) and their negative controls were obtained from Shanghai GenePharma Co., Ltd. Cell transfection for NCTC 1469 cells and RAW264.7 cells were performed using Lipofectamine 2000 regent (Invitrogen) based on the manual. Knock‐down or overexpression of TUG1 in mice was achieved through tail intravenous injection of LV vector (50 µL) for a week.