Mir control

For the stable transfection of anti-miR-361-5p, anti-miR-361-5p sequence were amplified from miRZip-361-5p construct (System Biosciences) and cloned into pSilencer4.1 system. BC cells were then transfected with the pSliencer vector containing the antisense sequence of miR-361-5p. The cells were selected by puromycin after 48 h transfection and then diluted. MiR-361-5p mimics, miR-control, FGFR1 siRNA, MMP-1 siRNA or siRNA negative control were purchased from Genepharma (China). FGFR1 and MMP-1 cDNA ORF Clone were purchased from Origene (Origene Technology). Transient transfections were performed by using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Cells were kept in medium containing 2% FBS for 48 h and then harvested and used.

The human osteosarcoma cell lines (including MG-63,U2OS,Saos-2 and SJSA-1), the normal bone cell line hFOB, and HEK293 cell line were purchased from the American Type Culture Collection (Manassas,VA, USA). These cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS). Cells were incubated at 37 °C in 5 % CO₂ humidity, and were passaged every 2–3 days. MiR-138 mimic (40 nM), miR-138 inhibitor (40 nM), DEC2-pcDNA3.1 (100 ng), DEC2 siRNA (100 ng) and the negative controls, including miR-Control (40 nM), pcDNA3.1 vector (100 ng), siRNA-Contrl (100 ng) were all purchased from GenePharma (Shanghai, China), and were transfected using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s instructions. About 48 h after transfection, the transfection efficiency was assessed. The cells could be used for subsequent analysis when the transfection efficiency was above 80 %.

MiR-375 mimic and miR-control were purchased from GenePharma (Shanghai, China). MiRNA transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA), according to the manufacturer’s instructions. The extent of overexpression was evaluated by qRT-PCR 24 h after transfection. MiR-375 mimic and miR-control lentiviruses were synthesised by Genechem (Shanghai, China). For stable overexpression of miR-375 in glioma cells, MiR-375 mimic or miR-control lentiviruses were added to cells, according to the manufacturer’s protocol. After 24 h, clones with stable expression were selected by incubating cells for 3 weeks with 5 μg/mL puromycin (Sigma, St Louis, USA) in complete medium containing 10% FBS. The sequences of miR-control and MiR-375 mimic were 5′-TTCTCCGAACGTGTCACGT-3′ and 5′-TTTGTTCGTTCGGCTCGCGTGA-3′, respectively.

miR‐155‐5p mimics, inhibitors, and a negative control (miR‐control) were purchased from GenePharma. In brief, 1 × 10⁶ of MSCs was plated in 10cm plate and then transiently transfected with 50 nM miR‐155‐5p mimics, inhibitors or miR‐control using Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) according to the protocol, respectively. The MSCs were incubated at a 37°C and 5% CO₂ incubator for 48 hr for transfection and then harvested for further experiments. The transfection experiments were repeated at least three times.

miR-control, miR-183-5p mimic and inhibitor were commercially acquired from GenePharma (Shanghai, China). Briefly, 1 × 10⁶ MSCs were plated on a 10-cm culture dish and transfected with 50 nM miR-183-5p mimic, inhibitor or miR-control using Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) according to the manufacturer’s instructions. Subsequently, MSCs were cultured at 37 °C in a 5% CO₂ incubator for 48 h and then harvested for further experiments.