Mir 34a

The cells were grown in 6-well dishes at a starting density of 1 × 10⁵ cells/well until a confluence of 85% in DMEM supplemented with 10% FBS; then, the media were replaced with DMEM 0.5% FBS for 6 h before transfection. Afterwards, synoviocytes were transfected with specific inhibitors of miR-34a, miR-146a, and miR-181a (Qiagen, Hilden, Germany), at the concentration of 50 nM, or with their relative negative controls siRNA (NC) (Qiagen, Hilden, Germany), at the concentration of 5 nM, in serum-free medium for a period of 24 h. Supernatants were removed and synoviocytes immediately harvested or incubated with visfatin (5 and 10 μg/mL) or resistin (50 and 100 ng/mL) for additional 24 h.

The methoxy polyethylene glycol-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-MPEG) and soybean phosphatidylcholine (SPC) were purchased from Lipoid GmbH (Germany); cholesterol, thiazolyl blue tetrazolium bromide (MTT), NAHCO₃ and fluorescent dye FAM (6-carboxyfluorescein) from Sigma-Aldrich Co (USA); and 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) from Avanti Polar Lipids (USA). Dulbecco's modified eagle medium (DMEM) low glucose, Glutamax® supplement, pyruvate, phosphate-buffered saline (PBS) tablets, penicillin/streptomycin/amphotericin B and trypsine-EDTA were purchased from Gibco (USA). Fetal bovine serum (FBS) was obtained from Invitrogen (USA). The human bone marrow-derived hMSC line S1939 was sourced from Royan Institute, Iran. The mature miRNA sequence was obtained from the miRBase database (http://www.mirbase.org). MicroRNA oligonucleotide miR-302a and miR-34a were prepared ready-to-use from Qiagen, Germany.

Cells were grown and maintained in 6-well dishes at a starting density of 1 × 10⁵ cells/well until they became 85% confluent in DMEM supplemented with 10% FBS, before replacement with 0.5% FBS for 6hr until transfection procedure. Afterwards, chondrocytes were transfected with miR-34a, miR-146a, and miR-181a specific inhibitors (Qiagen, Hilden, Germany), at the concentration of 50 nM, or with the negative controls siRNA (NC) (Qiagen, Hilden, Germany), at the concentration of 5 nM, in serum-free medium for 24~h. Media were removed and the cells were immediately collected or exposed to cycles of low sinusoidal or static continuous HP for a period of 3~h [29 (link),30 (link)].