Mir nc

The miR-376b and miR-376b-mimic expression plasmids and the negative control (miR-NC) were purchased from the Origene Company (Rockville, MD, USA). Plasmids were extracted using EndoFree Plasmid Giga kits (#12362; Qiagen GmbH, Hilden, Germany) from DH5α (Genewiz, Suzhou, China) Escherichia coli transformants and stored at −20°C until further use. The concentration was determined by measuring the A260/A280 ratio using an ND 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

miR-140-5p and miR-NC was obtained from Origene Technologies, Inc. The mature-miR-140-5p and miR-NC sequence was cloned into pCDHCMV-MCS-EF1-coGFP constructs (Invitrogen; Thermo Fisher Scientific, Inc.). For STAT3 overexpression, the coding sequences of STAT3 were amplified from cDNA isolated from RA FLS using a PCR kit (AP111-11; TransGen Biotech Co., Ltd.). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec. Sequences for primers were as follows: STAT3 forward, 5'-GACTTAGTCCCAGGTACT-3' and reverse, TTCAACTGACCTAGGACGTGGTCG-3'. The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11. Cells were transfected with pCDHCMV-MCS-EF1-coGFP-miR-140-5p and/or pcDNA3.1-SOX11 using Lipofectamine^® RNAiMAX/2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then collected at 48 h after transfection. All constructions were confirmed using plasmid DNA sequencing using the dideoxy chain-termination (Sanger) method with Applied Biosystems 3730XL (Genescript).