Pre mir control

HK-2 cells were cultured as previously described^{5 (link)} and reverse transfected with the pre-miR-control or pre-miR-146a (50 nM; Ambion, Foster City, CA, USA) using Lipofectamine (Qiagen, Germantown, MD, USA) in serum-free medium according to the manufacturer’s instructions. After transfection for 24 hours, the cells were washed and exposed to 50 ng/ml IL-1b for an additional 24 hours.

Human lung cancer cells (A549 cells) and mouse lung cancer cells (LLC cells) were purchased from the China Cell Culture Center (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO, Foster City, CA), all cells were incubated in a 5% CO₂ at 37°C in a water-saturated atmosphere. Anti-EPB41L3 and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Synthetic oligonucleotides, including pre-miR-223, anti-miR-223, and scrambled negative control (pre-miR-control and anti-miR-control), were purchased from Ambion (Austin, TX).

Synthetic pre-miR-93, anti-miR-93 and scrambled negative control RNAs (pre-miR-control and anti-miR-control) were purchased from Ambion (Austin, TX, USA). The cells were seeded onto 6-well plates or 60-mm dishes and transfected using Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. In each well, equal amounts of pre-miR-93, anti-miR-93 or scrambled negative control RNA were used. The cells were harvested at 24 h after transfection for quantitative RT-PCR analysis and Western blotting.

Pre-miR-203 and pre-miR-control were purchased from ThermoFischer Scientific, (Waltham, USA, #AM17100 pre-miR203a-3p ID assays PM10152 and #AM17111, respectively).
SiRNAs were purchased from ThermoFischer Scientific, Waltham, USA, with the following references: control siRNA, #12935-100; si-SRC(A), #1299001 ID HSS186080; si-SRC(B), #1299001 ID HSS186081, si-RAPGEF1(A), #1299001 ID HSS104431; si-RAPGEF1(B) #1299001 ID HSS104432.
NHK were cultured on a feeder layer of Swiss 3T3 fibroblasts as described previously^{75 (link)}. At 80% confluency they were trypsinized and transfected with pre-miRNA or siRNA at a final concentration of 10 nM, using lipofectamine RNAiMAX reagent (ThermoFischer Scientific, Waltham, USA) in Opti-MEM^® I Reduced Serum Medium (ThermoFischer Scientific, Waltham, USA). After one night of transfection, cells were grown at 37 °C, 5% CO₂, in KGM-Gold w/o Ca²⁺, phenol red free BulletKit (Lonza, Basel, Switzerland), prepared according to the manufacturer’s protocol but without gentamicin/amphotericin B and supplemented with calcium at a final concentration of 50 µM.

Cells were plated on 60 mm² plates at a density of 1 × 10⁶ cells per plate. When the cells were 70–80% confluent, they were transfected with pre-miR-21 or control pre-miR (Ambion, Austin, TX) using TransIT TKO transfection reagent (Mirus Bio, Madison, WI). Twenty-four hours post-transfection, cells were collected for use in all assays. Tissue inhibitor of metalloproteinase 3 (TIMP3)-specific small-interfering RNA (siRNA) and negative control constructs were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The sequences for each oligo are as follows: