Mir 29a 3p mimic

HEC-1A and Ishikawa cells in the logarithmic growth phase were harvested. These cells were prepared into cell suspension with serum-free RPMI-1640 medium (1 × 10⁶ cells/mL). A total of 1 mL cell suspension was seeded in 6-well plates. miR-29a-3p mimic and corresponding negative control (GenePharma, Shanghai, China) were respectively transfected into HEC-1A and Ishikawa cells (set as miR-29a-3p mimic group and miR-NC group, respectively). The full-length sequence of VEGFA was synthesized (GenePharma, Shanghai, China) and cloned into pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) according to the instructions. HEC-1A and Ishikawa cells were subjected to cotransfection with miR-29a-3p mimic and pcDNA3.1-VEGFA plasmid (named miR-29a-3p mimic + pcDNA-VEGFA group). All transfection operations were performed strictly in line with the instruction of Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells were kept at 37 °C, 5% CO₂ for 8 h. Subsequently, the residual liquid in each well was discarded. RPMI-1640 medium containing 10% FBS was used to culture cells for 48 h. HEC-1A and Ishikawa cells without transfection were cultured in RPMI-1640 medium containing 10% FBS (served as BLANK group).

Human VSMCs from umbilical artery from American-type culture collection (ATCC; Rockville, Maryland) were cultured in DMEM (11965092, Gibco, New York, USA) plus 10% fetal bovine serum (10270-106, Gibco) at 37°C with 5% CO₂. VSMCs were treated with different concentrations of ox-LDL (0, 25 μg/ml, 50 μg/ml, and 100 μg/ml). miR-29a-3p mimic, pcDNA3.1-TNFRSF1A, and their controls were purchased from the GenePharma (Shanghai, China). Cells were transfected with 30 nM oligonucleotides via Lipofectamine 2000 (11668019, Thermo, Shanghai, China).

We cultured human lung epithelial cells, BEAS-2B [29 (link)], in bronchial epithelial growth medium BulletKit at 37°C in a 5% CO₂ atmosphere. The cells were divided into 10 groups (Table 2). We subcultured the cells at a density of 1 × 10⁵/mL. After culturing the cells in the presence of 10 ng/mL TGF-β1 for 48 h, we transfected them with plasmid. The CRNDE NC-lncRNA, CRNDE si-lncRNA, NC mimic, miR-29a-3p mimic, oe-NC, and oe-MCL-1 shRNAs were supplied by GenePharma. We transferred BEAS-2B cells onto plates for transfections following the manufacturer's instructions.