Patients and tissue specimens
With the written informed consents obtained from each patient, a total of 60 knee cartilage tissues were collected from 2016 to 2018 in the Baoji Traditional Chinese Medicine Hospital, including 30 OA specimens from OA patients (mean age: 42.5 years old) and 30 normal specimens from emergency traumatic amputated patients (mean age: 34.6 years old). All patients with arthritis, including rheumatoid arthritis (RA), OA and septic arthritis, were excluded in control group. The samples were put into liquid nitrogen at once and stored for cryopreservation. This study was approved by the ethic committee of the Baoji Traditional Chinese Medicine Hospital.
Under aseptic conditions, the cartilage tissues were removed of fibrous connective tissues, and cut into small pieces. After washing with sterile phosphate-buffered saline (PBS), cartilage tissues were digested with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) for 30 min, and then with 0.2% collagenase Type II (Col2; Millipore Corp., Billerica, MA, USA) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) for 10 h at 37 oC. Subsequently, these cells were filtered with 40 mm filter, and then centrifuged and washed at least three times, last re-suspended in growth culture media supplemented with DMEM (Gibco), 10% fetal bovine serum (Gibco) and 100 U/ml penicillin and 100 µg/ml streptomycin solution (Invitrogen). The cells were cultured in humidified sterile air with 5% (v/v) CO2 at 37 oC, and the first passage chondrocytes were obtained for 10 days. The chondrocyte cells were treated with 0.25% trypsin (Invitrogen) for cell passage.
For OA cell model in primary chondrocytes, all experiments were depend on 1–3 passage cells exposed with IL-1β (Sigma-Aldrich, St. Louis, MO, USA). IL-1β was dissolved in ultrapure water to a storage concentration of 10 mg/ml according to the instruction. For IL-1β stimulation, chondrocytes were incubated in serum-free growth culture media containing IL-1β in a series of concentrations (0, 5, 10 and 20 ng/ml) for 24 h.
Cell counting kit-8 (CCK-8) assay
The viability of cells was determined according to CCK-8 (Dojindo Laboratories, Kumamoto, Japan) manufactures. In brief, the chondrocytes and transfected chondrocytes were treated with 0, 5, 10 and 20 ng/ml of IL-1β for 24 h. IL-1β-induced chondrocytes were seeded onto 96-well plate (Corning, NY, USA) at a density of 1 × 104 cells/well for 24 h. Cells were cultured with 20 µl CCK-8 solution (5 g/l) in PBS for another 2 h, and the optical density was measured at 450 nm using a microplate reader. Three independent wells were performed in each group.
IL-1β-induced chondrocytes were seeded onto 6-well plate (Corning) with 1 × 105 cells per well for 24 h and analyzed by Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, Shanghai, China) on flow cytometry. Apoptotic cells were labelled complying with the protocol. In short, the adherent were digested with 0.25% trypsin (Invitrogen) without EDTA, and harvested. Then, the cells were washed with ice PBS for twice, and re-suspended in 500 µl PBS. Next, 100 µl of cells in each group was stained in the binding buffer containing 5 µl FITC-Annexin V and 5 µl PI for 30 min at 25 oC in the dark. Fluorescence of labeled cells was analyzed on cytoFLEX LX flow cytometer (Beckman-Counter Electronics, Jiangsu, China) using CytExpert software.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was conducted to measure the concentration of IL-6 and TNF‐α released. Chondrocytes and transfected chondrocytes were seeded onto 24-well plates (Corning) for 24 h. After IL-1β stimulation, the culture supernatant was collected for estimation of inflammatory factors in line with the manufacturer’s instruction. The ELISA kits including human IL‐6 kit (ab46027), and human TNF‐α kit (ab46087) were purchased from Abcam (Cambrige, UK).
Real-time Quantitative PCR (RT-qPCR)
Total RNA from tissues and primary chondrocytes was isolated using TRIzol reagent (Life Technologies) following the protocol. Reverse transcription kit (Abcam) and/or Bestar QPCR RT Kit (DBI Bioscience, Germany) were utilized to reverse transcribe into cDNA depending on 300 ng of total RNA sample. The amplification of cDNA was performed using SYBR Premix Ex Taq Master Mix (Invitrogen) and/or Bestar SybrGreen qPCR MasterMix (DBI Bioscience) relying on special primers. The expression of miR-200a/b and FUT4 mRNA was analyzed on ABI PRISM 7900 Real-time PCR System (Applied Biosystems, Foster City, CA), and calculated according to the comparative threshold cycle value (2−ΔΔCt) method, compared with internal control GAPDH (for mRNA) or U6 small nuclear RNA (U6, for miRNA). All primers were synthesized by Ribobio (Guangzhou, China), including miR-200a: 5’-TAACACTGTCTGGTAACGATGT-3’ (sense) and 5’-CATCTTACCGGACAGTGCTGGA-3’ (antisense); miR-200b: 5’-GCTGCTGAATTCCATCTAATTTCCAAAAG-3’ (sense) and 5’-TATTATGGATCCGCCCCCAGGGCAATGGG-3’ (antisense); FUT4: 5’-CCGGCGAAGTTATCAAGGGTT-3’ (sense) and 5’-AAAGGAACAACTTTCCCCGA-3’ (antisense); U6: 5’-ACCCTGAGAAATACCCTCACAT-3’ (sense) and 5’-GACGACTGAGCCCCTGATG-3’ (antisense); GAPDH: 5’-GAAGATGGTGATGGGATTTC-3’ (sense) and 5’-GAAGGTGAAGGTCGGAGT-3’ (antisense). The reactions were performed in quadruplicate for each sample.
The primary chondrocytes were seeded onto 6-well plate (Corning) at density of 5 × 104/well prior to transfection for 24 h. For overexpression, the coding domain sequence (CDS) of FUT4 (NM_002033.3) was cloned into the multiple cloning site (MCS) of the pcDNA3.1 vector (Invitrogen); miR-200a mimic, miR-200b mimic and the negative control miR-NC mimic were purchased from GenePharma (Shanghai, China). Cell transfection with oligonucleotides (30 nM) or plasmids (2 µg) into primary chondrocytes was performed by lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. After transfection for 36 h, the cells were collected for functional analysis.
Dual-luciferase reporter assay
The putative target prediction of miR-200a/b and FUT4 was performed through DIANA Tools software. The potential binding sites of miR-200a/b on wild type of FUT4 3’UTR (FUT4-WT) were cloned by PCR method into plasmid pGL4-luciferase report vector (Promega, Madison, WI, USA), as well as the mutant of FUT4 3’UTR (FUT4-MUT). Cells were plated onto 24-well plate (Corning) at 1 × 104 cells/well, followed by co-transfection with 20 ng of FUT4-WT/MUT and 20 nM of either miR-200a/NC mimic or miR-200b/NC mimic in 293T cells for 48 h. All transfection groups were carried out in triplicate. Cells were collected to measure the relative luciferase activity using the dual-luciferase reporter assay system (Promega) in line with the manufacturer’s information.
RNA immunoprecipitation (RIP)
RIP was performed with chondrocyte extract after the primary chondrocytes were transfected with miR-200a mimic, miR-200b mimic or miR-NC mimic for 36 h. Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore) was chosen to obtain RIP-Ago2 and RIP-IgG through incubation with anti-Ago2 (ab32381, 1:25) or anti-IgG (ab2410, 1:100) at 4 °C for 16 h. Then, the enriched expression of FUT4 mRNA in the RIP samples was detected using RT-qPCR.
The total protein in IL-1β-treated chondrocytes was extracted by RIPA lysis buffer (Beyotime), and the concentration was determined using BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). 20 µg of proteins were separated on 8–12% SDS-PAGE and transferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, membranes carrying proteins were incubated with primary antibodies and HRP-conjugated secondary antibodies. β-actin was used as an internal standard to normalize protein level. The bands were visualized by ECL reagent (Millipore) according to the standard protocol. The primary antibodies were provided from Abcam and as follows: anti-FUT4 (ab181461, 1:500), anti-Collegen IIA1 (Col2a1; ab188570, 1:5000), anti-Aggrecan (ab52141, 1:100), and anti-β-actin (ab8227, 1:1000).
Data were presented as mean ± standard deviation (SD) from three independent experiments, and analyzed on Graphpad Prism 6.0 (GraphPad Software Inc., La Jolla, USA). Two tailed Student’s t test was used to calculate statistical significance between two groups test, and one-way analysis of variance was for multiple groups. Result with P value < 0.05 was considered to be statistically significant.