Mir 140 5p mimic

Healthy colon epithelial cell line (FHC) and CRC cell lines (HCT-116, SW480, Lovo, and SW620) were purchased from the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in DMEM medium supplemented with 10% FBS in a controlled environment of 5% CO₂ at 37 °C. Cell medium was replaced every 2 d. Before transfection, a total of 2.5 × 10⁴ cells per well were seeded onto 6-well plates and incubated for 24 h, then the culture medium was discarded and 100 nmol/L of CASC19 overexpressing plasmid (CASC19-p; Genechem, Shanghai, China), 200 nmol/L of siRNA mixture, 100 nmol/L of miR-140-5p mimic (Ribobio, Guangzhou, China), or 200 nmol/L of miR-140-5p inhibitor (Ribobio, Guangzhou, China) was used for cell transfection. The pcDNA 3.1 negative control (CASC19-p NC), pcNDA3.1-CASC19 (CASC19-p), CASC19 siRNAs (siCASC19), CEMIP siRNAs (siCEMIP), and pcNDA3.1-CEMIP (CEMIP-p) were purchased from Genechem (Shanghai, China). miR-140-5p mimic, miR-140-5p inhibitor, mimic NC, and inhibitor NC were obtained from Ribobio (Guangzhou, China).

The plasmids containing the wild-type Nrf2, wild-type Sirt2, mutant Nrf2, mutant Sirt2 and miR-140-5p mimic were purchased from Guangzhou RiboBio Co., Ltd. These were co-transfected into HUVECs using Lipofectamine 2000 24 h after transfection. Luciferase activity was determined using a Dual-Luciferase Reporter assay kit (Promega Corporation, Madison, WI, USA).

The lung cancer cell line A549 was purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.
For cell transfection, A549 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well. Briefly, a miR-140-5p mimic (5′-CAGUGGUUUUACCCUAUGGUAG-3′; 100 nM; Guangzhou RiboBio Co., Ltd., Guangzhou, China) or a negative control (NC) mimic (5′-CUCACCAAAAACCCUAUGGUAG-3′; 100 nM; Guangzhou RiboBio Co., Ltd.) and Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) were diluted in DMEM. Following this, the mixture was added to the 6-well plates to obtain a final concentration of 20 nmol/l of hsa-miR-140-5p mimics or miR-NC mimics, and subsequently incubated at 37°C for 48 h.