Samples of tissues
The synovial tissues from 25 RA patients were obtained during knee replacement surgery. RA was diagnosed according to the American College of Rheumatology criteria for classification of disease. Healthy synovial tissues from 16 patients with a traumatic knee condition were collected as the normal control. The study was approved by the Ethics Committee of The Third Clinical College of Wenzhou Medical University, Wenzhou People's Hospital, and all subjects provided informed consent.
Cell Culture And Transfection
The normal human FLSs and RA-FLSs were obtained from Cell Applications (San Diego, CA, U.S.A.). The frozen stocks were routinely resuscitated and cultured in DMEM medium containing 10% fetal bovine serum (FBS) with 5% CO2 in 37 °C. RA-FLSs cells were transfected with miR-6089 mimic, control mimic (miR-NC), lentiviral vectors CCR4 (siRNA1, siRNA2) and empty lentiviral vectors (Scramble) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions.
Cell Proliferation
Cell proliferation was detected by Cell Counting Kit-8 (CCK-8; Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China). Concisely, cells were harvested and seeded in a 96-well plate (2 × 103 cells/well). Following overnight incubation, 10 µL CCK-8 reagent was added to each well and the absorbance at 450 nm was determined after culturing for 24 h, 48 h and 72 h according to the manufacturer’s protocol.
Cell Apoptosis
The cells were collected, washed twice with cold 1 × PBS and resuspended in 100µL binding buffer, followed by incubation with Annexin V-FITC and propidium iodide for 15 min at room temperature in the dark. Next, 200µL of binding buffer was added, and the cells were analyzed by flow cytometry.
Luciferase Reporter Assay
According to TargetScan (http://www.targetscan.org) and MiRanda (http://www.microrna.org/microrna/home.do) databases, CCR4 was predicted as a putative target of miR-6089, which was verified using the dual luciferase reporter assay. Briefly, 2 × 104 cells/96 well in the logarithmic growth phase was cultured overnight, then 100 ng WT-CCR4-3’UTR, and MUT-CCR4-3’UTR luciferase reporter plasmid was co-transfected with 50 ng miR-6089 mimic or NC using Lipofectamine 3000, and the cells were cultured for 48 h. The relative luciferase activity was detected according to the dual luciferase assay kit instructions. Five replicates were tested per condition.
qRT–PCR analysis
Total RNA was extracted from collected cultured FLSs and tissue samples utilizing a Trizol reagent (Invitrogen, Carlsbad, CA), and the purity and concentration of RNA were determined with a spectrophotometer. The RNA was reversely transcribed into cDNA templates used for real-time RT-PCR, which was then performed on an 7500 real-time PCR system (ABI, Foster City, CA) utilizing a SYBR Premix kit (TaKaRa, Tokyo, Japan) to measure the relative expression of, miR-6089, and CCR4 according to the 2−ΔΔCt method. The primer sequences were as follows: miR-6089-F-5'-CCC CTT CCG TGC GCC AGT GG-3', miR-6089-R-5'-AGG CCG GGG TGG GC-3'; CCR4-F-5'-CCC ACG GAT ATA GCA GAC ACC-3', CCR4-R-5'-GTG CAA GGC TTG GGG ATA CT-3'; U6-F-5'-GCT TCG GCA GCA CAT ATA CTA AAA T-3', U6-R-5'-CGC TTC ACG AAT TTG CGT GTC AT-3'; GAPDH-F-5'-AGA AGG CTG GGG CTC ATT TG-3', GAPDH-R-5' AGG GGC CAT CCA CAG TCT TC-3'. U6 or GAPDH were used as the internal reference for miR-625 or CCR4, and the relative expression levels were analyzed using the 2−∆∆Ct method.
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Elisa Assays
The concentrations of MMP-1, TNF-α and IL-6 in the cell culture supernatant were detected using ELISA kits (Affymetrix, Santa Clara, CA, USA) following the manufacturer’s recommendations.
Western Blot Analysis
To prepare protein extracts, harvested cells were washed thrice with chilled PBS, and lysed with RIPA buffer supplemented with protease inhibitors. The extracted protein was quantified using the BCA method, and the cell lysates were diluted in 5 × loading buffer at the ratio of 1:4. After denaturing at 95 °C for 10 min, 30 µg protein per sample was subjected to SDS-PAGE. The protein bands were transferred to PVDF membranes and the latter were blocked with 5% skim milk at room temperature for 2 h. The blots were incubated overnight with primary antibodies against CCR4 (1:1000), Caspase-3(1:1000), Caspase-8(1:1000), Caspase-9 (1:1000), and GAPDH (1:5000) at 4 °C on a shaker. After washing thrice with TBST buffer, the blots were probed with HRP-conjugated secondary antibody (1:5000) for 1 h at room temperature, followed by 3 washes with TBST. ECL chromogenic substrate was used to visualize the bands, and the Image J software was used to determine the gray value of each band, and the content of each protein was calculated relative to that of GAPDH.
Statistical Analyses
SPSS 19.0 was used for statistical analysis. Descriptive statistics were used to determine the group means and standard deviations in the numerical data. Independent sample t-test was used to compare two groups, and one-way ANOVA along with Dunnett's or Bonferroni's test was used for multiple groups. The level of significance was set to p < 0.05.