Anti mir nc

The small interfering RNAs of circ_0051079 (si-circ_0051079#1, 5′-AGTCATCATTGCCAAGACTGT-3′; si-circ_0051079#2 5′-ATTGCCAAGACTGTGCCCTGT-3′; si-circ_0051079#3 5′-TCATTGCCAAGACTGTGCCCT-3′), specific miR-625-5p mimics (miR-625-5p, 5′-AGGGGGAAAGUUCUAUAGUCC-3′) and inhibitors (anti-miR-625-5p, 5′-GGACUAUAGAACUUUCCCCCU-3′), and controls (si-NC, miR-NC and anti-miR-NC) were synthesized by GenePharma (Shanghai, China). The coding sequence of TRIM66 was synthesized and introduced into the pcDNA 3.1 vector (vector) to build the overexpression plasmid of TRIM66 (TRIM66). The lentiviral expressing small hairpin RNA of circ_0051079 (sh-circ_0051079) or sh-NC were packaged using polybrene (GenePharma). Then, cells growing in 24-well plates were transfected with 20 pmol siRNA, 0.8 μg plasmid, 100 nM miRNA mimics or 200 nM miRNA inhibitors using Polyplus-transfection® (Illkirch, France), referring to the standard methods.

The primary chondrocyte were seeded into six-well plates (Corning, Inc.) at a density of 5x10⁴ cells/well for 24 h prior to transfection, and 293T cells were pre-seeded in 24-well plate at a density of 5x10³ cells/well. For overexpression purposes, the coding domain sequence of FUT4 was inserted into pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.), and equal amount of empty vector was used as negative control. miR-200b mimic and its negative control miR-NC mimic were purchased from Shanghai GenePharma Co., Ltd.. For knockdown purposes, anti-miR-200b and anti-miR-NC were provided by Shanghai GenePharma Co., Ltd.. Cell transfection was performed using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in serum-free DMEM at 37^˚C for 6 h, and the culture medium was replaced with complete cell culture DMEM medium for 48 h prior to further analysis. Briefly, 2 µg vectors and 30 nM oligonucleotides were transfected into cells in six-well plate, and 20 ng vectors and 20 nM oligonucleotides were transfected in a 24-well plate. After transfection for 48 h, chondrocytes were treated with 0 or 10 µg/ml LPS at 37^˚C for another 48 h.

The RNA oligonucleotides used in this study were purchased from GenePharma (Shanghai, China), including small interference (si) RNA against circ-OGDH (si-circ-OGDH#1 and si-circ-OGDH#2) and its matched negative control (si-NC), miR-615-5p mimic (miR-615-5p), negative control matched with miR mimic (miR-NC), miR-615-5p inhibitor (anti-miR-615-5p), and negative control corresponding miR inhibitor (anti-miR-NC). Overexpression plasmids of pCD5-ciR-circ-OGDH (circ-OGDH) and pcDNA-PDX1 (PDX1) were constructed using empty pCD5-ciR (Vector) (Geneseed, Guangzhou, China) and pcDNA (Thermo Fisher Scientific) vectors. Transient transfection was carried out using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) or Lipofectamine 3000 reagent (Thermo Fisher Scientific).

Plasmid LV3-pGLV-H1-GFP+Puro, with hsa-miR-320a mimics or hsa-miR-320a inhibitor, or their respective control oligonucleotides, namely miR-320a and miR-negative control (NC), and anti-miR-320a and anti-miR-NC, were purchased from GenePharma (Shanghai, China). Lentivirus transfection was performed as per the manufacturer's instructions to establish miR-320a-expressing stable clones (HepG2/miR-320a) and anti-miR-320a-expressing stable clones (HepG2/anti-miR320a) in HepG2 cells. The relative control clones (HepG2/miR-NC and HepG2/anti-miR-NC) were constructed by similar methods.