Mirna inhibitor

Primary OA chondrocytes were isolated and cultured according to a previous study [54 (link)–56 (link)]. Degenerated cartilage tissue samples were minced into pieces of less than 1 mm³, followed by digestion at 37 °C with 0.15% collagenase II (Invitrogen, Carlsbad, CA, USA) for 5 - 6 h with stirring every 20 min after 2 h. Chondrocytes were isolated after centrifugation and cultured in DMEM-F12 containing 10% fetal bovine serum (FBS) and antibiotics for 5 - 7 days before use. Isolated chondrocytes were identified by light microscopy, toluidine blue staining and type II collagen immunofluorescence staining (IF).
HRAS expression was achieved by transfection of si-HRAS or HRAS overexpressing vector (GeneCopoecia, Guangzhou, China); LINC00623 knockdown was achieved by transfection of sh1-LINC00623 or sh2-LINC00623 (GeneCopoecia). The modulation of miR-101 was achieved by transfection of miRNA mimics or miRNA inhibitors (Genepharma, Shanghai, China) with the help of Lipofectamine 2000 (Invitrogen).

Small interfering RNAs (siRNAs), miRNA mimics and their double-stranded negative controls (NC), and miRNA inhibitors and their single-stranded scrambled controls (NC inhibitor) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China).

The GFP-tagged LC3 cDNA expression construct was a gift from Dr. Noboru Mizushima (Tokyo Medical and Dental University, Tokyo, Japan). The miRNA mimics, miRNA inhibitors, ATG5 siRNA, ATG12 siRNA, pDsRed1 Atg12 were synthesized by GenePharma (Shanghai, China). Cells were transfected using Lipofectamine 2000 (Invitrogen, USA), according to the manufacturer's protocol.

The sequences for primers and oligonucleotides used in this study are provided in Table S1. The primers for qRT-PCR and plasmid construction were synthesized by TsingKe (Beijing, China). siRNAs, miRNAs, and miRNA inhibitors were synthesized by GenePharma (Shanghai, China). The probes used for FISH and circRNA precipitation were synthesized by General Biol (Anhui, China).

The function of miRNAs was investigated using synthetic, chemically modified, small RNAs that either mimicked (miRNA mimics) or inhibited (miRNA inhibitors) the activity of the specific miRNA in vitro. The BRCA1‐specific siRNAs, miRNA mimics (# B02003) and miRNA inhibitors (# B03001) were synthesized by GenePharm (Shanghai, China). The BRCA1‐siRNAs sequences were as follows: sense, 5’‐GGA AAC CUG UCU CCA CAA AGdTdT‐3’; anti‐sense, 5’‐CUU UGU GGA GAC AGG UUC CdTdT‐3’. The miRNA expression plasmids that contained the miRNA flanking sequence (~300nt), pBART17 and pBART19, were generated by inserting the PCR products into the pcDNA3.1 via HindIII and XhoI sites. The BRCA1 expression vector, pMH‐SFB‐BRCA1, was obtained from Addgene (plasmid #99394).⁴⁵In the experiment, 1‐2.5 µg of plasmids, 10 nmol/L of miRNA mimics and 20 nmol/L of siRNAs or miRNA inhibitors were used to transfect the cells in the 6‐well or 12‐well plate format. All transfections were performed using Lipofectamine 2000 (Invitrogen) with standard protocol unless otherwise specified. The stable miR‐BART‐expressing HK1 cells were selected with 200 µg/mL of G418 for 6 weeks.

miRNA inhibitors (GenePharma Co., China) were transfected into IMR-90 cells with Lipofectamine RNAiMAX (Invitrogen) at final concentrations of 100 nM according to the manufacturer's protocol.