Synthetic mirna mimics

The sequence from wild-type or mutants 3′-UTR of PRKCD and lincRNA-p21 containing the common miR-181 family binding site were synthesized and cloned downstream of the luciferase reporter gene into the pmirGLO vector (Promega, Madison, WI, USA) using the SacI and SalI restriction enzymes. For mutated 3′-UTRs, six or eight bases of each seed region were replaced with complementary bases. For the luciferase reporter assays, HEK293T cells were co-transfected with the indicated luciferase constructs with synthetic miRNA mimics (Ribobio, Guangzhou, China), and luciferase activity was measured using a dual reporter luciferase assay kit (Promega, Madison, WI, USA), according to the manufacturer’s protocol. Renilla luciferase activity was normalized to the corresponding firefly luciferase activity and plotted as a percentage of the controls.

Goat mammary epithelial cells (GMECs) were isolated from mammary gland tissue, and purified as previously reported [10 ]. These cells were treated in a lactogenic medium for 48 h to induce differentiation into secretary cells [11 (link), 12 (link)]. For cell experiments, GMECs were cultured in DMEM/F12 medium (Hyclone Laboratories, Beijing, China), containing 5 μg/mL insulin, 5 μg/mL hydrocortisone, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 ng/mL epidermal growth factor 1 (EGF-1, Gibco, Gaithersburg, MD, USA), and 10% fetal bovine serum (FBS, Biological Industries, BeitHaemek, Israel) in a humidified incubator with 5% CO₂ at 37 °C. Synthetic miRNA mimics were purchased from RiboBio (Guangzhou, Guangdong, China) and transfected into GMECs using the K2 transfection system (Biontex Laboratories GmbH, München, Germany) according to manufacturer’s instructions.