Mir nc

Recombinant lentivirus vector encoding antisense miR-34a (anti-miR-34a) or negative control (miR-NC) were constructed and purchased from GeneChem Co., Ltd. (Shanghai, China). Briefly, the oligonucleotides of antisense miR-34a inhibitor and NC were cloned into the lentivirus expression vector of hU6-MCS-CMV-EGFP (GeneChem Co., Ltd.). The recombinational and packaging vectors pHelper 1.0 and 2.0 (GeneChem Co., Ltd.) were co-transfected into 293T cells with Lipofectamine 2000 to produce viral particles of miR-34a antisense inhibitor and NC.

16HBE cells purchased from Procell (Wuhan, China) was cultured in RPMI-1640 (Invitrogen) that contained 10% fetal bovine serum (FBS; Invitrogen) in an incubator at 37°C with 5% CO₂. For CSE treatment, 16HBE cells were stimulated with various doses (0%, 1%, 2%, and 4%) of CSE, or CSE (2%) for indicated times (0 h, 6 h, 12 h, 24 h, and 36 h).
Small interfering RNA (siRNA) against circ_0040929 (si-circ_0040929) and corresponding control (si-NC), miR-515-5p mimics or inhibitor (miR-515-5p or anti-miR-515-5p) and corresponding control (miR-NC or anti-NC), IGFBP3 overexpression vector (IGFBP3) and corresponding control (vector) were all provided by Genechem (Shanghai, China). The sequences were as follows: si-circ_0040929 (5’-GACACAGCAGACGGTTGATGA-3’); si-NC (5’-GCGCGATAGCGCGAATATA-3’); miR-515-5p mimic (5’-UUCUCCAAAAGAAAGCACUUUCUG-3’); miR-NC (5’-ACUCUAUCUGCACGCUGACUU-3’); miR-1253 inhibitor (5’-CAGAAAGUGCUUUCUUUUGGAGAA-3’); anti-miR-NC (5’-CAGUACUUUUGUGUAGUACAA-3’). Lipofectamine 3000 Reagent (Invitrogen) was employed to transfect the oligonucleotide and/or vector into 16HBE cells.

For RNA transfection, 2×10⁵ PC-3 or PC-3M IE8 cells were cultured in six-well plates with antibiotic-free complete medium. Then the PC-3 cells were transfected with circMYLK or mock vector, and PC-3M IE8 cells were transfected with si-circMYLK or mock vector. Transfection was performed by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Then circMYLK and si-circMYLK sequences were synthesized by GenePharma (Shanghai, China). For miRNA transfection, the miR-29a mimics and miRNA controls (miR-NC) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). PC-3 cells were seeded in six-well plates (1×10⁵ cells/well) and were transfected with miR-29a mimics and miR-NC using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. After 48 hours, the cells were harvested for further experiments.