Mir 99a

H1299, HCC827, A549, PC9, H526, and DMS273 lung cancer cells and immortalized lung epithelial cells (BEAS-2B) were purchased from the Cell Bank (Shanghai, China). The cells were fostered in 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum, and placed in a 37℃, 5% CO₂ cell incubator. When the cell density reached 80–90% and was in the logarithmic growth phase, 0.25% trypsin was digested and passaged. According to the instructions, Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was obtained to transfect plasmids and small nucleic acids. Artificial sequences of si-THRIL (Catalogue number: 4,392,420, 5 nmol), si-NC (Catalogue number: 4,390,843, 5 nmol), miR-99a (Catalogue number: 4,464,084, 5 nmol), miR-NC (Catalogue number: 4,464,076, 5 nmol) were all purchased from Thermo Fisher (Waltham, MA, USA).

The luciferase experiments were performed in HEK293T cells. HEK293T cells were seeded at 1 × 10⁴ cells per well in a 96-well plate and were transfected the following day with Lipofectamine with the following molecules: the synthetic miRNA precursor miR-99a (ID: AM17100; Thermo Fisher Scientific), and negative control miR-C (ID: AM17110; Ambion), Cy3 (ID: AM17020; Thermo Fisher Scientific) and the pmirGLO plasmid (Promega Corporation, Madison, WI, USA) containing the luciferase reporter and also the Renilla gene (control) versus pmirGLO3′-UTR-E2F2 or pmirGLO3′-UTR-EMR2. The transfection efficiency was ∼95%, and luciferase activity was measured 48 h after transfection with the dual luciferase reporter assay as previously described.^{39 (link)} In each case, the miR-99a concentrations were measured by titrating the miR-99a with each pmirGLO3′-UTR mRNA construct to establish a dose–response relationship between 10 and 80 nM (data not shown). Results were conducted in triplicate in at least three independent experiments.