Introduction
Osteoarthritis (OA) is a joint degenerative disease, which is common in middle-aged and elderly people [
1,
2]. The pathological manifestations of OA are mainly including erosion of articular cartilage, marginal bone hyperplasia, and subchondral disease [
3,
4]. It is reported that there are more than 2700 million OA patients in the USA [
5,
6]. The prevalence rate of knee OA in people over 60 years old in China is as high as 49% [
7].
OA is characterized by increases in the pro-inflammatory cytokines and apoptosis of chondrocytes [
8]. Pathogenesis of OA is not yet fully understood and thus there is no effective treatment for OA [
9]. Current treatment strategies focus on controlling pain symptoms and delay joint degeneration [
10]. Therefore, there is an urgent need to develop new treatment options for OA.
Resveratrol (Res) is a non-flavonoid compound, which widely exist in the plant [
11]. Studies have confirmed that there are as many as 21 species of plants are rich of Res, such as grapes, peanuts, and cassia [
12]. Res was firstly isolated in the year of 1940 from the root of veratrumgrandiflorum. Large-scale experimental research found Res possess anti-aging, anti-oxidation, anti-inflammatory, anti-platelet aggregation, anti-atherosclerosis, anti-proliferation and anti-tumor activities, regulating cell apoptosis, and estrogen stimulation [
13‐
15].
Long non-coding RNAs (lncRNAs) belong to a novel category of non-coding RNA molecules harboring over 200 nucleotides [
16,
17]. Although lncRNAs exert no function of coding protein, they are extensively implicated in gene expression modulation, chromosome remodeling, and other physiological and pathological processing [
18]. In recent years, the expression profile and detailed functions of lncRNAs in osteoarthritis have received more and more interest and are being heavily studied [
19].
The aberrant expression of lncRNAs is frequently identified in osteoarthritis, implying that they may take part in the progression of osteoarthritis. An accumulating number of evidence has illuminated the anti-inflammation activity of lncRNAs, thus exerting important regulatory roles in osteoarthritis [
20].
MicroRNAs (miRNAs) are about 17–21 nucleotides in length and are defined as a group of single-strand non-coding RNA transcripts [
21,
22]. They post-transcriptionally control genes expression through complementary interacting with their target genes, consequently blocking the translations and protein accumulation [
23]. LncRNAs and miRNAs can interact with each other, thus affecting the metabolic activities of cells. In the fields of competitive endogenous RNA (ceRNA) theory, lncRNAs harbor miRNA response elements and can competitively bind to miRNAs, consequently weakening the repressive effect of miRNAs on target mRNAs and regulating genes at posttranscriptional level [
24]. Therefore, the exploration of lncRNAs in osteoarthritis may be vital for the discovery of novel diagnostic and therapeutic targets.
In this study, we performed RNA sequencing about control and resveratrol-treated chondrocytes and then construct lncRNA-miRNA-mRNA network. Therefore, the present study may advance the understanding of the underlying molecular mechanisms of OA and may contribute to the diagnosis and treatment of OA.
Material and methods
Chondrocytes isolated and cultured
Chondrocytes were isolated as previously described [
25]. The cartilage tissues from patients that underwent total knee arthroplasty were cut into 3–5 mm
3 slices. Then, the slices were digested with 0.2% collagenase type II to isolate primary chondrocytes. Then, chondrocytes were washed with PBS for three times and then incubated in a humidified incubator (37 °C and 5% CO
2) for 24 h. To mimic the OA inflammatory condition, IL-1β (10 ng/ml, 2 h) was chosen to treat human chondrocytes directly. To study the treatment of resveratrol, the chondrocytes were prematurely exposed to 25 μM resveratrol and then treated with IL-1β (10 ng/ml, 2 h).
RNA sequencing
RNA from chondrocytes treated with IL-1β or IL-1β + resveratrol was extracted with TRIzol by standard methods. Concentration of total RNA, RIN value, 28S/18S, and fragment size were detected with Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). Agarose gel electrophoresis was used to detect RNA integrity. The cDNA library was established using the RNA-Seq Library Preparation Kit (Illumina) following the manufacturer’s protocol. Sequencing was performed on the Illumina HiSeq4000 sequencing platforms at Majorbio Biotech Co., Ltd.
Normalization was performed using median normalization. Differentially expressed genes (DEGs) analysis was performed with R software, using package DESeq2. Heatmap and volcano plot visualizations were performed using the R package “pheatmap” and “ggplot2,” respectively. Gene Ontology (GO) and KEGG pathway enrichment were performed using DAVID software (
https://david-d.ncifcrf.gov/). lncRNA–miRNA and miRNA–mRNA pairs were used to construct a lncRNA–miRNA–mRNA network using Cytoscape v.3.6.1 software. The binding interaction between LINC00654 and miR-210-5p was predicted utilizing StarBase 3.0 (
http://starbase.sysu.edu.cn/). Three bioinformatics tools, namely TargetScan (
http://www.targetscan.org/), miRDB (
http://mirdb.org/), and StarBase 3.0, were used to forecast the possible target genes of miR-210-5p.
RNA preparations and quantitative reverse transcription–polymerase chain reaction (qRT-PCR)
The extraction of total RNA from collected samples or cells was carried out utilizing total RNA Purification Kit (Norgen Biotek Corp., Belmont, CA, USA). RNA was subjected to a NanoDrop™ 2000 Spectrophotometer (Invitrogen; Thermo Fisher Scientific, Inc.) for the determination of RNA purity and concentration. The first-strand complementary DNA was prepared from total RNA with a PrimeScript™ RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Tokyo, Japan). Then, PCR amplification was implemented with a TB Green Premix Ex Taq (Takara Biotechnology Co.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was taken as the control for target gene expression. U6 small nuclear RNA served as the internal reference for miRNA. All genes expression was analyzed by 2−ΔΔCt method.
Dual luciferase reporter assay
LINC00654 or OGFRL1 containing the putative binding sites of miR-210-5p was obtained from Genepharma and cloned into the vectors (pGL6; Beyotime) for establishment of reporter vectors LINC00654 (WT/MT) or OGFRL1 (WT/MT). LINC00654 (WT/MT) or OGFRL1 (WT/MT) was transfected into chondrocytes together with vector-control (NC) or miR-210-5p mimic using Lipofectamine 2000 (Thermo Fisher Scientific). The result was analyzed by the Dual-Glo Luciferase Assay System (Promega).
Apoptosis
Briefly, chondrocytes (3 × 105/ml) were seeded in a 6-well plate and incubated for 24 h. The cells were then incubated with IL-1β (10 ng/ml) at 37 °C. Cell suspension was collected in 15 mL centrifugation and centrifuge at 1000 rpm for 5 min in the tube. Then, 1 mL PBS was added to resuspend the cells and then centrifuge again at 1000 rpm for 5 min. Five microliters of Annexin V-FITC was added and incubated at room temperature in the dark for 10 min. Five microliters of PI was then incubated at room temperature for 5 min in the dark. Cell apoptosis was examined by a flow cytometer (BD Biosciences) and then was analyzed using Flowjo version 7.6 software (Flowjo LLC).
Statistical analysis
Nine biological replicates were performed for each experiment and mean values were calculated. Differences among multiple cell transfection groups were analyzed using ANOVA (one-way) and Tukey’s test. P < 0.05 was statistically significant.
Discussion
This is the first study that aimed to identify the differentially expressed lncRNAs, miRNAs, and mRNAs between resveratrol and control. High-throughput RNA sequencing was performed and we selected one lncRNA to validate. We construct lncRNA-miRNA-mRNA network to explain the mechanism of resveratrol in protecting against osteoarthritis. Previous study has identified that resveratrol alleviates the IL-1β-induced chondrocytes injury through the NF-κB signaling pathway [
26]. Xu et al. [
27] revealed that resveratrol inhibits the development of obesity-related osteoarthritis via the TLR4 and PI3K/Akt signaling pathways. Further studies confirmed their findings and identified that resveratrol alleviating obesity-related osteoarthritis via alleviating JAK2/STAT3 signaling pathway, which independent of SOCS3. Zhang et al. [
28] found that resveratrol could prevent osteoarthritis progression via the MALAT1/miR-9/NF-κB signaling pathway. Further in vivo study found that resveratrol ameliorates inflammatory damage and protects against osteoarthritis in a rat model of osteoarthritis [
29]. However, there is no study about the resveratrol for regulating lncRNA-miRNA-mRNA regulation network.
With the improvement of high-throughput sequencing, more lncRNAs were identified. A total of 1016 differentially expressed lncRNAs were identified between resveratrol-treated chondrocytes.
A substantial body of evidences have implicated that lncRNAs participated into the osteoarthritis progression. Luo et al. [
30] found that knockdown of lncRNA MFI2-AS1 inhibits lipopolysaccharide-induced osteoarthritis progression by miR-130a-3p/TCF4. Huang et al. [
31] revealed that lncRNA DILC is downregulated in osteoarthritis and regulates IL-6 expression in chondrocytes. Tang et al. [
32] found that lncRNA TUG1 promotes osteoarthritis-induced degradation of chondrocyte extracellular matrix via miR-195/MMP-13 axis. In this study, we further performed RT-PCR and identify the expression of LINC00654 in control and resveratrol-treated chondrocytes. Further study identified that LINC00654 has putative targeting sites with miR-210-5p through bioinformatic analysis. Wu et al. [
33] performed a miRNA sequencing and identified miR-210-5p is highly enriched in OA sclerotic subchondral bone osteoblast exosomes. Gu et al. [
34] found that oral resveratrol prevents osteoarthritis progression in C57BL/6J mice fed a high-fat diet. These researches verified that resveratrol could be a potential therapeutic target for osteoarthritis.
Conclusion
In sum, the present study for the first time detected the differential expressed lncRNAs involved in resveratrol-treated chondrocytes via employing bioinformatic methods. An lncRNA-associated ceRNA network was established and hub lncRNA, such as LINC00654, were identified. Functional enrichments disclosed that resveratrol could regulate LINC00654 expression, which could compete with OGFRL1 to combine with miR-210-5p.
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