Mir 146a

miRNA isolation was performed with a miRNeasy kit (Qiagen, Venlo, Netherlands) according to manufacturer instructions. Stem-loop RT-qPCR miRNA expression profiling of 760 microRNAs was done as previously described [32] (link), [33] (link). Raw expression values were normalized using the global mean normalization strategy [32] (link). Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). MicroRNA specific QPCR primer sets against human mir-146a (MS00003535), mir-491-5p (MS00031899), mir-146b-5p (MS00003542), mir-26b-5p (MS00003234), mir-150-5p (MS00003577), mir-330-3p (MS00031738), mir-195 (MS00003703), mir-184(MS00003640), mir-125a-5p (MS00003423) were purchased from Qiagen (Venlo, Netherlands). For microRNA target and pathway prediction, miRWalk software was applied [34] (link). Correlation of mRNA and microRNA transcription profiles of MM1S and MM1R cells either non-treated or treated with Dex, or following mock transfection or transfection with mir-150-5p mimetic was performed with IPA software.

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.