OA cartilage was obtained from the weight-bearing surface of the femoral condyle and tibial plateau of 4 female and 1 male patients diagnosed with OA (using the American College of Rheumatology classification criteria), aged between 57 and 69 years old with an average age of 63.4±2.42, and undergoing total knee arthroplasty between October 2017 to March 2018 in the Department of Sports Medicine of the First Affiliated Hospital of Kunming Medical University (Kunming, China) (31,32). Written informed consent was obtained from all patients, and the present study was approved by the Ethics Committee at the First Affiliated Hospital of Kunming Medical University (Kunming, China).

Chondrocytes were digested with 0.15% collagenase and cultured in high glucose Dulbecco's Modified Eagle Medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and 100 µg/ml streptomycin. Culture medium was filtered to remove bacteria using a 0.22 µm microfilter. The first generation chondrocytes were used and divided into two groups: Treatment and control. To mimic the osteoarthritic environment of the knee joint, each group was treated with 100 ng/ml SDF-1 (PeproTech, Rocky Hill, NJ, USA). The treatment group was pretreated with 1 µM TN14003 (Scilight Biotechnology, LLC, Beijing, China) for 2 h prior to the addition of SDF-1. Each group of chondrocytes was incubated at 37°C and 5% CO₂ for 2 days.

For miRNA screening, total RNA was isolated from cartilage tissues with or without TN14003 treatment, purified and prepared using the Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany; cat. no. 74106) according to manufacturer's protocol. The integrity and quantity of samples were determined via Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). miRNAs were isolated from total RNA using the All-in-One™ miRNA reverse transcription quantitative polymerase chain reaction (RT-qPCR) Detection kit (GeneCopoeia, Inc., Rockville, MD, USA), according to the manufacturer's protocol.

To calibrate the initial results, differentially expressed miRNAs were identified using miProfile™ human inflammatory miRNA qPCR Arrays (GC08017K18014P; GeneCopoeia, Inc.). The chondrocyte samples were isolated from 5 patients with OA and the validation was performed using samples with or without TN14003 treatment. Each well contained a forward primer for the mature miRNA sequence, and a universal adaptor reverse primer cross-linked to the 96-well plate. The primers for measuring miRNA expression were designed as summarized in Table I, and the qPCR was performed using 20 µl reaction volumes containing 1 µl reverse transcription product and SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The amplification conditions were as follows: Pre-incubation at 50°C for 2 min, enzyme activation at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 10 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. Detection was performed using an ABI 7500 instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.), and 2^−ΔΔCq was used to calculate the relative expression of miRNAs, as previously described (33). Alterations with a fold-change >2 and P<0.05 were considered to be differentially expressed.

A total of 7 differentially expressed miRNAs (miR-146a-5p, miR-221-3p, miR-126-3p, miR-185-5p, miR-155-5p, miR-124-3p and miR-130a-3p) were identified (Fig. 1). The targets of these miRNAs were predicted using miRDB (34) (http://mirdb.org/miRDB/), miRTarBase (35) (http://mirtarbase.mbc.nctu.edu.tw/), miRWalk (36) (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) and TargetScan (37) (http://www.targetscan.org/). Genes targeted by miRNAs were analyzed by Gene Ontology (GO) analysis (38) (http://geneontology.org/page/go-enrichment-analysis) and the Database for Annotation, Visualization and Integrated Discovery pathway analysis (39) (http://www.genome.jp/) to compile gene annotation terms and involved signaling pathways. Finally, miRNA-target networks were constructed to visually enrich the gene dataset. The enrichment result was visualized by Enrichment Map as a plugin of Cytoscape version 3.2.0 (40). The revised P-value was characterized by the false discovery rate (FDR) to avoid false positivity, and an FDR<0.05 was used as the cut-off value.

Chondrocytes were seeded in 6-well plates at 5×10⁴ cells per well and incubated at 37°C for 24 h. Chondrocytes were separately transfected with miR-146a-5p mimics, miR-146a-5p inhibitors and negative controls (NC; GeneCopoeia, Inc.) at a 100 nM concentration for 48 h using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc, MA, USA), according to the protocol of the manufacturer. The sequences for miR-146a-5p mimics, miR-146a-5p inhibitors and the corresponding NC were as follows: miR-146a-5p mimic, 5′-UGAGAACUGAAUUCCAUGGGUU-3′ (sense) and 5′-CCCAUGGAAUUCAGUUCUCAUU-3′ (antisense); mimics NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGGAATT-3′ (antisense); miR-146a-5p inhibitor, 5′-AACCCAUGGAAUUCAGUUCUCA-3′; inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA-3′. The chondrocytes was cultured for 48 h after transfection and then used for subsequent experiments.

Total RNA was extracted from cartilage tissues using TRIzol^® regent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was transcribed into cDNA using the RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc). cDNA (40 µg/µl) was used as a template for amplification of CXCR4, type II collagen (Col II), aggrecan (ACAN) and MMP-3 genes, and β-actin served as an internal reference. SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for RT-qPCR analysis. The amplification conditions were as follows: Pre-incubation at 50°C for 2 min, enzyme activation at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 10 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. All primers (Table II) were obtained from the NCBI database and designed by Premiers Express Software v1.0 (BioTools Incorporated, Edmonton, AB, Canada). Reactions for each sample were performed in at least three independent experiments. Cycle threshold values were measured and data were calculated the by 2^−ΔΔCq method (33).

Total protein was extracted from cells following lysis with radioimmunoprecipitation assay buffer and quantified by the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Total protein (30 µg) was separated via electrophoresis on 10% SDS-PAGE gels prior to transfer to polyvinylidene fluoride membranes. Membranes were probed with primary antibodies in TBS with 5% non-fat milk. The antibodies included were anti-CXCR4 (LifeSpan BioSciences, Inc., Seattle, WA, USA; 1:1,000 dilution; cat. no. LS-B2160-0.05), anti-Col II (Abcam, Cambridge, UK; 1:1,000 dilution; cat. no. ab188570) and anti-β-actin (Abcam, Cambridge, UK; 1:5,000 dilution; cat. no. ab8227), and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Abcam, Cambridge, UK; 1:10,000 dilution; cat. no. ab97051) was used as the secondary antibody. Proteins of interest were visualized using enhanced chemiluminescent reagent (Thermo Fisher Scientific, Inc.). The band intensities were quantified by densitometry using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA) (41). Experiments were repeated at least 3 times.

All quantitative data were analyzed with SPSS 18.0 (SPSS, Inc. Chicago, IL, USA) and presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance with the Least-Significant Difference correction to determine differences between groups. P<0.05 were considered to indicate a statistically significant difference.

To evaluate the effects of TN14003 on chondrocytes, OA patients-derived chondrocytes were treated with SDF-1 alone or SDF-1 + TN14003 for 48 h. Cells were harvested to investigate the alteration of miRNA profile upon the inhibition of CXCR4/SDF-1 axis by TN14003. There were 7 differentially expressed miRNAs identified in cartilage samples via microarray analysis (Table III). Among these miRNAs, 5 miRNAs (miR-146a-5p, miR-221-3p, miR-126-3p, miR-185-5p and miR-155-5p) were significantly upregulated, and 2 miRNAs (miR-124-3p and miR-130a-3p) were significantly downregulated in the treatment group compared with the control group (Fig. 1A). In order to understand the mechanism of these miRNA alterations and consequently their involvement in OA treatments, the miRWalk database and Cytoscape software was used to analyze the miRNA-mRNA interactions through their visualization as a network (Fig. 1B). While the interactions between miRNAs and potential targets were built, a sequence diagram was performed to reveal the importance of targets. The importance of targets was determined according to the number of connections between each gene and miRNA and other genes in the miRNA-target network (Fig. 1C). As demonstrated in the network map, the miRNAs hsa-miR-146a-5p, hsa-miR-221-3p, hsa-miR-126-3p and hsa-miR-185-5p exhibited direct interaction with the mRNA CXCR4 (Fig. 1B). Among all the potential targets, EGFR, GRB2, CBL, CXCR4, ESR1, PTPN11, SHC1 and SOS1 were the targeted mRNAs with highest importance scores (Fig. 1C).

To validate the differentiated expression of the 7 miRNAs identified from the initial screening, the levels of these miRNAs in control-treated and TN14003-treated chondrocytes were measured by RT-qPCR assays. Compared with the initial microarray results, the expression levels of miR-146a-5p, miR-126-3p and miR-124-3p were validated by the RT-qPCR approach: As indicated in Fig. 1A, the expression of both miR-146a-5p and miR-126-3p was upregulated, while the expression of miR-124-3p was downregulated; the results of RT-qPCR analysis presented in Fig. 2A confirmed this observation. However, other miRNAs that did not exhibit the same changes, or exhibited no statistical differences in expression were excluded from subsequent analyses (Fig. 2A). Notably, miR-146a-5p was upregulated >3-fold. A previous study suggested an association between miR-146a-5p and OA, as evidenced by its >2-fold increased expression compared with healthy controls (42). Besides, accumulating data indicate that miR-146a-5p is a representative miRNA known to be associated with OA (43,44). Therefore, the present study focused on miR-146a-5p and its targets to delineate their associations with OA treatments by TN14003.

GO analysis indicated that miR-146a-5p and its targets were primarily grouped into ‘receptor regulator activity’ or ‘nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) activity’ in the Molecular Functions category, into ‘lipopolysaccharide-mediated signaling pathways’ or ‘cellular response to ketones’ in the Biological Processes category, and into ‘secondary lysosome’, ‘sperm midpiece’ or ‘endosomes’ in the Cellular Components category (Fig. 2B). Enriched pathways of miR-146a-5p and its targets were primarily involved in the ‘chemokine signaling pathway’ or the ‘Toll-like receptor signaling pathway’ (Fig. 3). These results indicated that these identified inflammation-associated GO terms and pathways are important to the roles of the SDF-1/CXCR4 axis in the pathogenesis of OA.

To additionally reveal the roles of miR-146a-5p in the process of OA development, the mimic and inhibitor of miR-146a-5p were designed to examine the effects of molecular manipulation of miR-146a-5p on OA-associated molecules. First, an RT-qPCR assay was performed to verify the effect of cell transfection and successful upregulation and downregulation of miR-146a-5p expression. As demonstrated in Fig. 4, the level of miR-146a-5p was significantly increased ~12-fold compared with the mimic control in chondrocytes transfected with the miR-146a-5p mimic, and markedly downregulated to ~20% of the inhibitor control when the miR-146a-5p inhibitor was transfected. As hypothesized, there were no significant changes in miR-146a-5p expression between chondrocytes that were treated with SDF-1 only and transfected with negative controls (Fig. 4).

Next, RT-qPCR was performed to measure the expression of cartilage degeneration-associated factors (including Col II, ACAN, CXCR4 and MMP-3) following transfection of the chondrocytes with the mimic and inhibitor of miR-146a-5p, and corresponding NCs. As indicated in Fig. 5A and B, in the chondrocytes transfected with the miR-146a-5p mimic, the expression levels of Col II and ACAN were significantly increased. However, there was no significant difference in Col II expression between chondrocytes transfected with the inhibitor NC and the miR-146a-5p inhibitor. The expression levels of ACAN decreased markedly in the chondrocytes transfected with the miR-146a-5p inhibitor. In contrast, as indicated in Fig. 5C and D, the mRNA expression levels of CXCR4 and MMP-3 decreased in the chondrocytes following transfection with the miR-146a-5p mimic, and increased following transfection with the miR-146a-5p inhibitor. These results demonstrate a positive association between miR-146a-5p expression and the mRNA expression levels of Col II and ACAN, and a negative association between miR-146a-5p expression and the mRNA expression of CXCR4 and MMP-3.

As CXCR4 was confirmed to be a target of miR-146a-5p, and Col II is the principal component of cartilage ECM, effects of miR-146a-5p expression on the protein levels of these 2 factors were additionally assessed. Consistent with the alteration of mRNA expression, expression of the CXCR4 protein was downregulated as a result of miR-146a-5p mimic transfection (Fig. 6A and B). Concomitantly, the protein level of CXCR4 was significantly upregulated following miR-146a-5p inhibitor transfection. As hypothesized, the opposite pattern of expression was observed for Col II protein in chondrocytes transfected with the mimic or inhibitor of miR-146a-5p (Fig. 6A and C). Taken together, these results highlight the critical roles of miR-146a-5p on regulating the expression of cartilage degeneration-associated factors.