Analyzing miRNA Expression in 293T Cells

The human 293T cell line was acquired from Jishishengming (Beijing, China) and grown in a humidified atmosphere at 37°C with 5% CO₂ using MEM medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Corning, Shanghai, China) and 1% penicillin and streptomycin antibiotics (Gibco, Shanghai, China). The culture medium was replaced every other day. PcamiR1, its analogue miRNA (has-mir-365a-5p), and a control miRNA were designed and supplied by Jishishengming (Beijing, China). Their sequences are listed in Table F in S1 Text. The SiLentFect lipid reagent (Bio-Rad, California, USA) was used for transfection, and the experiments were performed as per Vinther [80 (link)]. Protein was extracted from the water-treated cells or transfected cells after 48 h and detected by western-blotting.