Mir 210

To confirm the results from miRNA-Seq, we selected some miRNAs of interest to measure using PCR. Total RNA was isolated from AMSCs by the mirVana PARIS total RNA isolation kit (Life Technologies, Carlsbad, CA, USA, Cat# AM1556) according to the kit protocol. RNA concentrations were measured by a NanoDrop Spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc.). A fixed volume of 5 μL of RNA elute at 1 ng/uL was reverse transcribed by using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Cat# 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan Universal Master Mix (Cat# 4440038), 7.67 μL of H2O and 1 μL of primers, including miR-21, miR-146a, miR-155, and miR-210 (Life Technologies, Cat# 000397, 001097, 002623, and 000512 respectively) to make up a 20 μL reaction. RNU6B (Life Technology Cat# 001093) was included in the assay as reference control. Real-time PCR was carried out on an Applied Biosystems (Foster City, CA, USA) ViiA7 Real-Time PCR system at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold changes of miRNA levels in hypoxia relative to normoxia were calculated using the 2^−ΔΔCt method.

Isolated cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and 1% antibiotic–antimycotic solution (Nacalai Tesque, Kyoto, Japan). Inner meniscus cells (5 × 10⁴) or synovial cells (5 × 10⁴) were seeded into the wells of 12-well plates and incubated for between 3 and 24 hours. The cells were transfected with miR-210 (20 nM) or silencer negative control RNA (20 nM; Life Technologies) using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies).