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MiR-18a-3p improves cartilage matrix remodeling and inhibits inflammation in osteoarthritis by suppressing PDP1

MiR-18a-3p improves cartilage matrix remodeling and inhibits inflammation in osteoarthritis by... Osteoarthritis (OA) is a degenerative disease characterized by synovial inflammation. MiR-18a-3p was reported to be downregulated in knee anterior cruciate ligament of OA patients. In the present study, the specific functions and mechanism of miR-18a-3p in OA were explored. An in vitro model of OA was established using 10 ng/ml IL-1β to treat ATDC5 cells, and medial meniscus instability surgery was performed on Wistar rats to establish in vivo rat model of OA. RT-qPCR revealed that miR-18a-3p was downregulated in IL-1β-stimulated ATDC5 cells. MiR-18a-3p overexpres- sion inhibited secretion of inflammatory cytokines and concentration of matrix metalloproteinases, as shown by ELISA and western blotting. The binding relation between miR-18a-3p and pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) was detected by luciferase reporter assays. MiR-18a-3p targeted PDP1 and negatively regulated PDP1 expression. Results of rescue assays revealed that PDP1 upregulation reserved the suppressive effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1β-stimulated ATDC5 cells. H&E staining was used to observe pathological changes of synovial tissues in the knee joint of Wistar rats. Safranin O-fast green/hematoxylin was used to stain cartilage samples of knee joints. MiR-18a-3p overexpression sup- pressed OA progression in vivo. Overall, miR-18a-3p improves cartilage matrix remodeling and suppresses inflamma- tion in OA by targeting PDP1. Keywords: OA, miR-18a-3p, PDP1, Chondrocyte, Inflammation Introduction through osteoarthritic remodeling, cartilage erosion, and Osteoarthritis (OA) is an articular joint disease with high fibrillation [5]. In recent years, mounting medical expen - morbidity in the elderly people [1]. From 2005 to 2015, ditures used for OA treatment have brought a burden on approximately 200 million of people were afflicted with OA patients and the whole society [6]. Therefore, there is OA, 2% of whom suffered from physical disability [2]. The an urgent need to look for an effective therapy of OA. etiological factors of OA include obesity, heredity, cardio- The main pathological changes of OA are articular car - vascular diseases, aging, genetic factors and joint injury tilage degeneration, inflammation of synovitis, and sec - [3]. OA is attributed to premature onset of OA after joint ondary bone hyperplasia [7, 8]. Cartilage, a connective injury [4]. Clinically, the manifestation of OA includes tissue with high specialization, is composed of chondro- narrowing of the joint space, emergence of osteophytes cytes and extracellular matrix [9]. Chondrocytes synthe- size extracellular matrix to maintain the structural and functional integrity of the cartilage [10]. However, chon- drocytes are difficult to maintain cartilage homeostasis in *Correspondence: yamadix@163.com Department of Orthopedics, Changzhou Cancer Hospital Affiliated response to OA stimulation [11]. Chondrocytes convert to Soochow University, No.68 Honghe Road, Xinbei District, to catabolic cells that secrete matrix-degrading enzymes, Changzhou 213000, Jiangsu, China © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 2 of 10 such as matrix metalloproteinases (MMPs), and carti- into OA rats (n = 8 rats) or sham-operated rats (n = 8 lage degeneration was induced when catabolic-degrad- rats). ing effects overwhelm the anabolic-protective function in OA chondrocytes [10]. Chondrocyte catabolism can Establishment of OA animal model be stimulated by inflammatory cytokines including A total of 32 Wistar rats (male, 8-week-old) were tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) obtained from the Jackson Laboratory (USA) and were and prostaglandin E2 (PGE2) [10]. TNF-α, for example, randomly divided into four groups: sham + AAV-miR- can induce the expression of proteases such as MMP1, NC, sham + AAV-miR-18a-3p, OA + AAV-miR-NC MMP2, MMP3, MMP8, MMP9, MMP14, ADAMTS-4 and OA + AAV-miR-18a-3p. Each group has 8 rats. All and ADAMTS-5 [12]. Many previous studies also verified rats were kept in a pathogen-free environment, and all that inflammation in the synovitis is a key factor leading operations were performed under isoflurane anesthesia, to OA progression [10, 13, 14]. with all efforts made to minimize suffering. In OA group, MicroRNAs (miRNAs) are noncoding RNAs with rats were performed medial meniscus instability surgery 22–25 nucleotides in length [15]. The involvement of on the right knee after anesthetization with isoflurane miRNAs in OA was reported in numerous studies [16]. to induce joint instability and posttraumatic OA. Rats in For example, miR-485-3p regulates the Notch2/NF-κB sham group received similar surgical procedure without pathway to inhibit apoptosis and promote chondrocyte removing the ligament or meniscus. After surgery, AAV- proliferation in OA [17]. MiR-132 upregulation facili- miR-NC and AAV-miR-18a-3p were injected into OA tates the proliferative ability and suppresses the apop- rats or sham-operated rats. After 8  weeks, all rats were tosis of chondrocytes in OA by modulating the PTEN/ euthanized. Then, synovial tissues, cartilage tissues and PI3K/AKT signaling pathway [18]. Overexpressed miR- blood samples were collected. All animal experiments 144-3p reduces the numbers of IL-1β-positive cells in were approved by the Animal Care and Use Committee synovial tissue to ameliorate OA pathogenesis [19]. MiR- of Changzhou Cancer Hospital Affiliated to Soochow 18a-3p was reported to be downregulated in knee ante- University (Jiangsu, China) and performed in accordance rior cruciate ligament of OA patients [20]. Additionally, with the committee’s guidelines. miR-18a-3p targets HOXA1 to induce apoptosis of chon- drocytes in OA [21]. Although miR-18a-3p can partici- Cell culture and treatment pate in OA pathogenesis, its effects on cartilage matrix The chondrogenic cell line ATDC5 (Riken BioResource remodeling and inflammation in OA are still unknown. Center, Tsukuba, Japan) was incubated in DMEM/F12 In this study, we explored the functional role of miR- (Gibco, Massachusetts, USA) supplemented with 10% 18a-3p in cartilage matrix deposition and inflammatory FBS (Gibco) at 37  °C with 10% C O . ATDC5 cells were response of chondrocytes in OA and investigated the treated with 10  ng/ml IL-1β (Roche Diagnostics, Ger- underlying mechanism of miR-18a-3p. The study may many) for 2  h and cells with DMSO (0.1%) treatment help us understand the molecular mechanism underlying served as the control. OA development and provide insight into effective thera - pies for OA patients. Cell transfection MiR-18a-3p mimics was used to overexpress miR-18a-3p and the negative control (NC) was named as miR-NC. Material and methods Full-length sequence of pyruvate dehydrogenase phos- Bioinformatic analysis phatase catalytic subunit 1 (PDP1) was subcloned into The potential target mRNAs of miR-18a-3p were pre - the pcDNA3.1 vector to elevate PDP1 expression with dicted by miRDB (http:// mirdb. org/) (screening con- pcDNA3.1 as a control. All plasmids were constructed dition: score rank ≤ 8), and the specific binding site by Genepharma (Shanghai, China). Cell transfection was between miR-18a-3p and PDP1 3′UTR was predicted by conducted using Lipofectamine 2000 (Invitrogen, USA) Targetscan (http:// www. targe tscan. org/). according to the manufacturer’s protocols, and the trans- fection efficiency was examined using reverse transcrip - tion quantitative polymerase chain reaction (RT-qPCR) Adeno‑associated virus (AAV) injection after 48 h. To overexpress miR-18a-3p in joint tissues, the AAV serotype 2 containing miR-18a-3p mimics or miR-NC were constructed and packaged by Hanbio Company Hematoxylin–eosin (H&E) staining (Shanghai, China). After surgery, AAV-miR-18a-3p First, synovial tissues collected from sham-operated con- (1 × 10 PFUs in a total volume of 5  μl) and the control trol rats and OA rats were fixed with paraformaldehyde, AAV-miR-NC (5  μl) were intra-articular (IA)-injected dehydrated, paraffin-embedded, and sliced into sections. F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 3 of 10 After that, H&E was used to stain the tissues. A micro- RT‑qPCR scope was used to observe pathological changes of syno- After transfection, the total RNA was extracted from vial tissues. The standard of pathological score was set as ATDC5 cells and tissues of OA rats using TRIzol rea- previous described [22]. Higher score represented greater gent (Invitrogen). Total RNA was reverse transcribed joint injury severity. into cDNA utilizing a PrimeScript product RT reagent Kit (Takara, Kyoto, Japan). Then, cDNA was amplified by polymerase chain reaction (PCR). U6 was the internal Safranin O/fast green staining reference of miR-18a-3p and GAPDH was the internal Knee joints of sham-operated control rats and OA rats reference of mRNAs. The results were analyzed using the were deparaffinized in xylene, hydrated with gradient −ΔΔCt 2 method. The primer sequences are presented in ethanol, and stained with safranin-O/fast green. Scoring Additional file 1: Table S1. criteria was introduced in this study [23]. Statistical analysis Enzyme‑linked immunosorbent assay (ELISA) The data are presented as the mean ± standard deviation For in vitro experiments, ATDC5 cells (2 × 10  cells/well) and statistics were analyzed by GraphPad Prism software were pretreated with different vectors in 6-well plates for 6.0 (La Jolla, CA, USA). Student’s t test was utilized for 2  h and then stimulated with IL-1β. For in  vivo experi- difference comparison between two groups, and one-way ments, blood samples were collected from rats infected analysis of variance followed by Tukey’s post hoc analysis with AAV. Then, serum was obtained after blood sam - were used for evaluation of differences among multiple ples were centrifuged at 6000×g for 15  min at 4  °C. The groups. A value of p < 0.05 was considered statistically concentrations of inflammatory cytokines (IL-6, IL-8 significant. and PGE2) in cells or serum were assessed using cor- responding ELISA kits (Elabscience Biotechnology, Results Wuhan, China). Absorbance values were read at 450 nm MiR‑18a‑3p overexpression improves cartilage matrix in a microplate reader (BioTek synergy HT, Bedfordshire, remodeling and inflammation in an in vitro model of OA UK). First, we used IL-1β to stimulate ATDC5 cells to con- struct an in  vitro model of OA. After IL-1β treatment, Western blotting we observed that miR-18a-3p expression in ATDC5 cells The concentration of total proteins obtained from was downregulated (Fig. 1A). miR-18a-3p expression was ATDC5 cells and tissues of OA rats was determined by increased in ATDC5 cells after transfection with miR- a BCA assay kit (Beyotime, Shanghai, China). Next, the 18a-3p mimic (Fig.  1B). Then, secretion of inflammatory proteins were loaded and separated by 8% SDS-PAGE. cytokines including IL-8, IL-6 and PGE2 was examined Then, the proteins were transferred onto PVDF mem - by ELISA. The results showed that IL-1β increased the branes and blocked with 5% skim milk for 1  h. After- release of these inflammatory cytokines in ATDC5 cells, wards, the membranes were incubated with primary and miR-18a-3p overexpression reversed IL-1β-induced antibodies including anti-MMP2 (ab92536; 1:1000), anti- increase in cytokines (Fig.  1C–E). Meanwhile, the pro- MMP3 (ab52915; 1:1000), anti-MMP9 (ab76003; 1:1000), tein levels of matrix metalloproteinases (MMP2, MMP3, anti-PDP1 (ab198261; 1:200), and anti-β-actin (ab8226; and MMP9) were increased in ATDC5 cells after IL-1β 1:1000) at 4  °C overnight and then with secondary anti- treatment compared with those in control groups and body horseradish peroxidase-labeled IgG (ab6721; were decreased in IL-1β-stimulated ATDC5 cells silenc- 1:2000) at 4 °C for 2 h. Protein bands were visualized by ing miR-18a-3p compared with those in cells transfected BeyoECL Plus (Beyotime) and analyzed using Image Lab with miR-NC (Fig.  1F). All these data suggested that 3.0 (Invitrogen). an in  vitro model of OA was successfully established. In addition, miR-18a-3p is downregulated in IL-1β- Luciferase reporter assay stimulated ATDC5 cells, and miR-18a-3p overexpression The wild-type (Wt) PDP1 3′UTR fragment contain - suppress levels of matrix metalloproteinases and proin- ing binding site of miR-18a-3p was inserted into the flammatory cytokines in the in vitro model of OA. pmirGLO vector (Promega, Madison, WI) to generate the luciferase reporter PDP1-Wt. PDP1-Mut was con- MiR‑18a‑3p targets PDP1 structed by insertion of mutated PDP1 3′UTR into the To explore the mechanism of miR-18a-3p in IL-1β- pmirGLO vector. PDP1 3′UTR Wt/Mut were transfected stimulated ATDC5 cells, potential target genes of miR- into ATDC5 cells with miR-18a-3p mimic or miR-NC 18a-3p were predicted by miRDB and the first eight genes using Lipofectamine 2000 (Invitrogen). Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 4 of 10 Fig. 1 MiR-18a-3p overexpression improves cartilage matrix remodeling and inflammation in IL-1β-stimulated ATDC5 cells. A MiR-18a-3p expression in ATDC5 cells with or without IL-1β treatment was detected by RT-qPCR. B Transfection efficiency of miR-18a-3p mimics in IL-1β-stimulated ATDC5 cells was detected by RT-qPCR. After IL-1β treatment or miR-18a-3p overexpression, C–E the secretion of inflammatory cytokines IL-8, IL-6 and PGE2 in ATDC5 cells was examined by ELISA. F Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) were examined by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001 (SNX8, FNDC5, EFNA1, ZBBX, ADCY1, PDP1, UBE2Z level in ATDC5 cells (Fig. 2E, F). Therefore, it can be con - and THSD7B) were identified (Fig.  2A). After overex- cluded that PDP1 is targeted by miR-18a-3p in ATDC5 pressing miR-18a-3p, only PDP1 was downregulated in cells. ATDC5 cells among these candidates, as shown by RT- qPCR (Fig. 2B). The binding site of miR-18a-3p on PDP1 PDP1 upregulation reserves the inhibitory effect 3′UTR was predicted by TargetScan, and the binding site of miR‑18a‑3p overexpression on levels of inflammatory is highly conserved among species (Fig.  2C). Luciferase cytokines and matrix metalloproteinases in the in vitro reporter assay was used to validate the binding relation model of OA between miR-18a-3p and PDP1 3′UTR, which revealed To identify the role of the miR-18a-3p/PDP1 regulatory that miR-18a-3p upregulation significantly decreased the axis in IL-1β-treated ATDC5 cells, rescue assays were luciferase activity of PDP1 3′UTR-Wt rather than that of performed. MiR-18a-3p overexpression downregulated PDP1 3′UTR-Mut (Fig.  2D). In addition, overexpressing PDP1 mRNA and protein levels in IL-1β-stimulated miR-18a-3p contributed to the decrease in PDP1 protein ATDC5 cells, while PDP1 upregulation reversed this F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 5 of 10 Fig. 2 MiR-18a-3p targets PDP1. A Potential target genes (SNX8, FNDC5, EFNA1, ZBBX, ADCY1, PDP1, UBE2Z and THSD7B) of miR-18a-3p were predicted by miRDB. B The expression of potential target genes in ATDC5 cells transfected with miR-18a-3p mimics or miR-NC was detected by RT-qPCR. C The binding site of miR-18a-3p for PDP1 3′UTR was predicted by TargetScan. D Luciferase reporter assay was used to validate the binding relation between miR-18a-3p and PDP1 3′UTR in ATDC5 cells. E, F PDP1 protein level in ATDC5 cells after overexpressing miR-18a-3p was examined by western blotting. **p < 0.01 trend (Fig.  3A, B). MiR-18a-3p-induced decrease in effect of miR-18a-3p overexpression on levels of matrix inflammatory cytokines (IL-8, IL-6 and PGE2) was metalloproteinases and proinflammatory cytokines in reversed by PDP1 elevation in IL-1β-stimulated ATDC5 IL-1β-stimulated ATDC5 cells. cells (Fig. 3C–E). Furthermore, miR-18a-3p upregulation led to reduction of matrix metalloproteinases (MMP2, MiR‑18a‑3p overexpression inhibits OA progression in vivo MMP3 and MMP9) in IL-1β-stimulated ATDC5 cells, To further validate the functions of miR-18a-3p in OA which was offset by PDP1 overexpression (Fig.  3F). Taken pathogenesis, in  vivo experiments were also conducted. together, PDP1 upregulation reserves the suppressive A total of 32 Wistar rats were randomly divided into Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 6 of 10 Fig. 3 PDP1 upregulation reserves the inhibitory effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1β-stimulated ATDC5 cells. A, B PDP1 protein level in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 was examined by western blotting. C–E The secretion of inflammatory cytokines IL-8, IL-6 and PGE2 in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 was examined by ELISA. F Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 were examined by western blotting. **p < 0.01, ***p < 0.001 IL-8, IL-6 and PGE2 were higher in OA model rats than four groups (n = 8/group): sham + AAV-miR-NC; in sham-operated rats, and miR-18a-3p overexpres- sham + AAV-miR-18a-3p; OA + AAV-miR-NC and sion inhibited the secretion of inflammatory cytokines OA + AAV-miR-18a-3p. According to the results from (Fig.  4C). The increase in matrix metalloproteinases H&E staining, miR-18a-3p overexpression attenuated (MMP2, MMP3 and MMP9) in OA rats was reversed the pathological changes of synovial tissues in OA rats, after upregulating miR-18a-3p (Fig.  4D, E). Finally, miR- including alleviated proliferation of synovial cells and 18a-3p expression in cartilage tissues was detected using reduced inflammatory cells (Fig.  4A). In addition, miR- RT-qPCR. After AAV-miR-18a-3p infection, miR-18a-3p 18a-3p elevation significantly reduced cartilage deg - was overexpressed in cartilage tissues of sham-operated radation of OA rats according to results of Safranin O/ rats (Fig.  4F). Compared with rats in the sham group, Fast Green Staining (Fig.  4B). Moreover, serum levels of (See figure on next page.) Fig. 4 MiR-18a-3p overexpression inhibits OA progression in vivo. A H&E staining was used to observe pathological changes of synovial tissues in the knee joint of sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p. B Safranin O-fast green/hematoxylin was used to stain histologic samples of knee joints of sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p. C Serum levels of IL-8, IL-6 and PGE2 in sham-operated control rats and OA rats infected with AAV-miR-NC or AAV-miR-18a-3p were examined by ELISA. D, E Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) in sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p were examined by western blotting F miR-18a-3p expression in cartilage tissues of rats of four groups was detected using RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001 F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 7 of 10 Fig. 4 (See legend on previous page.) Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 8 of 10 OA model rats exhibited decreased miR-18a-3p expres- act as a protective role in OA progression; in the study sion (Fig.  4F). Additionally, AAV-miR-18a-3p success- revealing the enhancer role of miR-18a-5p in TNFα- fully upregulated miR-18a-3p in cartilage tissues of OA induced cartilage destruction and chronic inflammation model rats (Fig.  4F). Overall, miR-18a-3p expression [31], NF-κB is an inducer of miR-18a-5p and NF-κB acti- levels in vivo were consistent with results of miR-18a-3p vation is known to be involved in many chronic inflam - expression detection in IL-1β-stimulated ATDC5 cells. matory diseases [32]. In the current study, the target Overall, miR-18a-3p elevation inhibits OA progression gene PDP1 is identified to promote inflammation in in vivo. many diseases [33–35]. u Th s, we suspected that the dif - ferent role of upstream or downstream genes may affect the functions of miR-18a-3p and miR-18a-5p in arthritis. Discussion Here, we also investigated the functions of miR-18a-3p OA is a degenerative disease that poses a threat to health in ATDC5 cells after IL-1β treatment. We found that condition of humans [24]. IL-1β can induce articular IL-1β-stimulated the increase in concentration of inflam - chondrocytes to produce cytokines and chemokines, matory cytokines and matrix metalloproteinases was thus leading to inflammation [25]. IL-1β upregulates attenuated by miR-18a-3p upregulation in ATDC5 cells. MMPs to damage articular cartilage structure and unbal- More importantly, miR-18a-3p alleviated the pathologi- ance metabolism [26]. Thus, IL-1β overexpression is a cal changes of OA rats in in vivo experiments. In conclu- hallmark of OA progression [27]. In this study, IL-1β sion, miR-18a-3p inhibits OA progression by improving was used to treat ATDC5 cells to stimulate the in  vitro cartilage matrix remodeling and suppressing inflamma - pathological environment of OA. After IL-1β stimula- tion (Fig. 5). tion, the secretion of inflammatory cytokines (IL-8, IL-6 MiRNAs are gene regulators that directly bind to and PGE2) and the concentration of matrix metallopro- 3′UTR of their mRNAs to degrade transcripts or sup- teinases (MMP2, MMP3 and MMP9) were increased in press protein translation [36, 37]. In the current study, ATDC5 cells. The results suggested that an in vitro model PDP1 was identified as a target gene of miR-18a-3p and of OA was successfully established. negatively regulated by miR-18a-3p. PDP1 is also called Accumulating studies have revealed that miRNAs PDH, PDP, PDPC, PPM2A, PPM2C [38]. PDP1 overex- are involved in OA progression [16, 28, 29]. In the cur- pression leads to exhaustion of hematopoietic stem cells rent study, miR-18a-3p was downregulated in an in vitro to increase the occurrence of myeloproliferative disor- model of OA, suggesting that miR-18a-3p participated der [39]. Many studies revealed that induction of PDP1 in the pathogenesis of OA. MiR-18a-3p was reported to be downregulated in the knee anterior cruciate ligament of OA patients [20], which is consistent with our results. However, in another study, miR-18a-3p was reported to be upregulated in chondrocytes isolated from mouse and human cartilage tissues and directly target downstream gene homeobox A1 to facilitate chondrocyte apoptosis [21], which is opposite to our findings and the results of miRNA sequencing done by Bin et al. [20]. Furthermore, miR-18a, also termed miR-18a-5p, is derived from the same pri-miRNA as miR-18a-3p. MiR-18a-5p is over- expressed in primary Sjögren’s syndrome, a disease that can cause muscle and joint pain and muscle weakness [30]. MiR-18a-5p increased the expression of MMP1, inflammatory cytokines and chemoattractant proteins in rheumatoid arthritis synovial fibroblasts through NF-κB dependent manner after TNFα stimulation [31]. The pre - vious study concluded that miR-18a-5p is an enhancer of TNFα-induced cartilage destruction and chronic inflam - mation in the joint [31]. These published articles revealed Fig. 5 As shown in the diagram, IL-1β downregulates miR-18a-3p that miR-18a-3p and miR-18a-5p might play a different in ATDC5 cells. miR-18a-3p directly targets PDP1, and PDP1 role in arthritis. Moreover, the functions of a miRNA promotes the concentration of inflammatory cytokines and matrix metalloproteinase. Thus, miR-18a-3p suppresses levels of might be affected by its upstream or downstream genes. inflammatory cytokines and matrix metalloproteinase by targeting In the study revealing the miR-18a-3p/HOXA1 axis in PDP1 in IL-1β-stimulated ATDC5 cells OA [21], HOAX1 inhibits chondrocyte apoptosis and F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 9 of 10 Authors’ contributions expression is Notch-dependent, and PDP1 is involved XF and HX were the main designers of this study. XF, JL, YW and HX performed in the activation of proinflammatory macrophages [33] the experiments and analyzed the data. HX drafted the manuscript. All authors and mouse hepatic macrophages [34]. PDP1 was also read and approved the final manuscript. reported to function as an integral signaling target for Funding proinflammatory stimuli (formyl-methionyl-leucyl-phe - None. nylalanine and leukotriene B4) and anti-inflammatory Availability of data and materials mediators (lipoxins) to regulate the activation of poly- The data underlying this article will be shared on reasonable request to the morphonuclear neutrophils in neutrophil proinflam - corresponding author. matory responses [35]. Similarly, in this study, PDP1 upregulation reverses the inhibitory impact of miR- Declarations 18a-3p upregulation on inflammatory cytokines in IL-1β- Ethics approval and consent to participate stimulated ATDC5 cells. The association between PDP1 All institutional and national guidelines for the care and use of laboratory and MMPs has not been clearly reported yet. Here, we animals were followed. discovered that overexpressing PDP1 rescued the sup- Consent for publication pressive effect on protein levels of MMP2, MMP3 and Not applicable. MMP9 in IL-1β-stimulated ATDC5 cells. All these results suggested that PDP1 promotes inflammatory response in Competing interests The authors have no competing interests. OA development, and miR-18a-3p inhibits inflammation in OA by targeting PDP1. Received: 15 September 2021 Accepted: 19 January 2022 In the published article, NF-κB is an inducer of miR- 18a in TNFα-induced rheumatoid arthritis synovial fibroblasts [31]. Additionally, NF-κB activation is the principal pathway of Notch signaling pathways, and PDP1 is a target of the Notch pathway and is activated References by Notch [33, 34]. The Notch pathway exerts a proinflam - 1. Boesen M, Ellegaard K, Henriksen M, Gudbergsen H, Hansen P, Bliddal H et al (2017) Osteoarthritis year in review 2016: imaging. Osteoarthr Cartil matory effect on OA [40]. Thus, we suspected that NF-κB 25(2):216–226 or Notch signaling is associated with PDP1 expression in 2. Vos T, Allen C, Arora M, Barber RM, Bhutta ZA, Brown A, Carter A, Casey IL-1β-stimulated ATDC5 cells. However, whether NF-κB DC, Charlson FJ, Chen AZ, Coggeshall M (2016) Global, regional, and national incidence, prevalence, and years lived with disability for 310 dis- or Notch signaling is responsible for the downregula- eases and injuries, 1990–2015: a systematic analysis for the Global Burden tion of miR-18a-3p in IL-1β-stimulated ATDC5 cells and of Disease Study 2015. Lancet 388(10053):1545–1602 whether NF-κB or Notch signaling is involved in the miR- 3. Chen D, Shen J, Zhao W, Wang T, Han L, Hamilton JL et al (2017) Osteoarthritis: toward a comprehensive understanding of pathological 18a-3p/PDP1 axis still needs to be further investigated. mechanism. Bone Res 5:16044 There are some limitations in our study. First, the pre - 4. Duan X, Cai L, Pham CTN, Abu-Amer Y, Pan H, Brophy RH et al (2021) Intra- sent study lacked human biopsy examination to implicate articular silencing of periostin via nanoparticle-based siRNA ameliorates post-traumatic osteoarthritis in mice. Arthritis Rheumatol. https:// doi. org/ the clinical relevance of our preclinical data. Second, the 10. 1002/ art. 41794 effects of PDP1 on chondrocytes and OA rats were not 5. Everhart JS, Jones MH, Yalcin S, Reinke EK, Huston LJ, Andrish JT et al evaluated. 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MiR-18a-3p improves cartilage matrix remodeling and inhibits inflammation in osteoarthritis by suppressing PDP1

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Abstract

Osteoarthritis (OA) is a degenerative disease characterized by synovial inflammation. MiR-18a-3p was reported to be downregulated in knee anterior cruciate ligament of OA patients. In the present study, the specific functions and mechanism of miR-18a-3p in OA were explored. An in vitro model of OA was established using 10 ng/ml IL-1β to treat ATDC5 cells, and medial meniscus instability surgery was performed on Wistar rats to establish in vivo rat model of OA. RT-qPCR revealed that miR-18a-3p was downregulated in IL-1β-stimulated ATDC5 cells. MiR-18a-3p overexpres- sion inhibited secretion of inflammatory cytokines and concentration of matrix metalloproteinases, as shown by ELISA and western blotting. The binding relation between miR-18a-3p and pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) was detected by luciferase reporter assays. MiR-18a-3p targeted PDP1 and negatively regulated PDP1 expression. Results of rescue assays revealed that PDP1 upregulation reserved the suppressive effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1β-stimulated ATDC5 cells. H&E staining was used to observe pathological changes of synovial tissues in the knee joint of Wistar rats. Safranin O-fast green/hematoxylin was used to stain cartilage samples of knee joints. MiR-18a-3p overexpression sup- pressed OA progression in vivo. Overall, miR-18a-3p improves cartilage matrix remodeling and suppresses inflamma- tion in OA by targeting PDP1. Keywords: OA, miR-18a-3p, PDP1, Chondrocyte, Inflammation Introduction through osteoarthritic remodeling, cartilage erosion, and Osteoarthritis (OA) is an articular joint disease with high fibrillation [5]. In recent years, mounting medical expen - morbidity in the elderly people [1]. From 2005 to 2015, ditures used for OA treatment have brought a burden on approximately 200 million of people were afflicted with OA patients and the whole society [6]. Therefore, there is OA, 2% of whom suffered from physical disability [2]. The an urgent need to look for an effective therapy of OA. etiological factors of OA include obesity, heredity, cardio- The main pathological changes of OA are articular car - vascular diseases, aging, genetic factors and joint injury tilage degeneration, inflammation of synovitis, and sec - [3]. OA is attributed to premature onset of OA after joint ondary bone hyperplasia [7, 8]. Cartilage, a connective injury [4]. Clinically, the manifestation of OA includes tissue with high specialization, is composed of chondro- narrowing of the joint space, emergence of osteophytes cytes and extracellular matrix [9]. Chondrocytes synthe- size extracellular matrix to maintain the structural and functional integrity of the cartilage [10]. However, chon- drocytes are difficult to maintain cartilage homeostasis in *Correspondence: yamadix@163.com Department of Orthopedics, Changzhou Cancer Hospital Affiliated response to OA stimulation [11]. Chondrocytes convert to Soochow University, No.68 Honghe Road, Xinbei District, to catabolic cells that secrete matrix-degrading enzymes, Changzhou 213000, Jiangsu, China © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 2 of 10 such as matrix metalloproteinases (MMPs), and carti- into OA rats (n = 8 rats) or sham-operated rats (n = 8 lage degeneration was induced when catabolic-degrad- rats). ing effects overwhelm the anabolic-protective function in OA chondrocytes [10]. Chondrocyte catabolism can Establishment of OA animal model be stimulated by inflammatory cytokines including A total of 32 Wistar rats (male, 8-week-old) were tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) obtained from the Jackson Laboratory (USA) and were and prostaglandin E2 (PGE2) [10]. TNF-α, for example, randomly divided into four groups: sham + AAV-miR- can induce the expression of proteases such as MMP1, NC, sham + AAV-miR-18a-3p, OA + AAV-miR-NC MMP2, MMP3, MMP8, MMP9, MMP14, ADAMTS-4 and OA + AAV-miR-18a-3p. Each group has 8 rats. All and ADAMTS-5 [12]. Many previous studies also verified rats were kept in a pathogen-free environment, and all that inflammation in the synovitis is a key factor leading operations were performed under isoflurane anesthesia, to OA progression [10, 13, 14]. with all efforts made to minimize suffering. In OA group, MicroRNAs (miRNAs) are noncoding RNAs with rats were performed medial meniscus instability surgery 22–25 nucleotides in length [15]. The involvement of on the right knee after anesthetization with isoflurane miRNAs in OA was reported in numerous studies [16]. to induce joint instability and posttraumatic OA. Rats in For example, miR-485-3p regulates the Notch2/NF-κB sham group received similar surgical procedure without pathway to inhibit apoptosis and promote chondrocyte removing the ligament or meniscus. After surgery, AAV- proliferation in OA [17]. MiR-132 upregulation facili- miR-NC and AAV-miR-18a-3p were injected into OA tates the proliferative ability and suppresses the apop- rats or sham-operated rats. After 8  weeks, all rats were tosis of chondrocytes in OA by modulating the PTEN/ euthanized. Then, synovial tissues, cartilage tissues and PI3K/AKT signaling pathway [18]. Overexpressed miR- blood samples were collected. All animal experiments 144-3p reduces the numbers of IL-1β-positive cells in were approved by the Animal Care and Use Committee synovial tissue to ameliorate OA pathogenesis [19]. MiR- of Changzhou Cancer Hospital Affiliated to Soochow 18a-3p was reported to be downregulated in knee ante- University (Jiangsu, China) and performed in accordance rior cruciate ligament of OA patients [20]. Additionally, with the committee’s guidelines. miR-18a-3p targets HOXA1 to induce apoptosis of chon- drocytes in OA [21]. Although miR-18a-3p can partici- Cell culture and treatment pate in OA pathogenesis, its effects on cartilage matrix The chondrogenic cell line ATDC5 (Riken BioResource remodeling and inflammation in OA are still unknown. Center, Tsukuba, Japan) was incubated in DMEM/F12 In this study, we explored the functional role of miR- (Gibco, Massachusetts, USA) supplemented with 10% 18a-3p in cartilage matrix deposition and inflammatory FBS (Gibco) at 37  °C with 10% C O . ATDC5 cells were response of chondrocytes in OA and investigated the treated with 10  ng/ml IL-1β (Roche Diagnostics, Ger- underlying mechanism of miR-18a-3p. The study may many) for 2  h and cells with DMSO (0.1%) treatment help us understand the molecular mechanism underlying served as the control. OA development and provide insight into effective thera - pies for OA patients. Cell transfection MiR-18a-3p mimics was used to overexpress miR-18a-3p and the negative control (NC) was named as miR-NC. Material and methods Full-length sequence of pyruvate dehydrogenase phos- Bioinformatic analysis phatase catalytic subunit 1 (PDP1) was subcloned into The potential target mRNAs of miR-18a-3p were pre - the pcDNA3.1 vector to elevate PDP1 expression with dicted by miRDB (http:// mirdb. org/) (screening con- pcDNA3.1 as a control. All plasmids were constructed dition: score rank ≤ 8), and the specific binding site by Genepharma (Shanghai, China). Cell transfection was between miR-18a-3p and PDP1 3′UTR was predicted by conducted using Lipofectamine 2000 (Invitrogen, USA) Targetscan (http:// www. targe tscan. org/). according to the manufacturer’s protocols, and the trans- fection efficiency was examined using reverse transcrip - tion quantitative polymerase chain reaction (RT-qPCR) Adeno‑associated virus (AAV) injection after 48 h. To overexpress miR-18a-3p in joint tissues, the AAV serotype 2 containing miR-18a-3p mimics or miR-NC were constructed and packaged by Hanbio Company Hematoxylin–eosin (H&E) staining (Shanghai, China). After surgery, AAV-miR-18a-3p First, synovial tissues collected from sham-operated con- (1 × 10 PFUs in a total volume of 5  μl) and the control trol rats and OA rats were fixed with paraformaldehyde, AAV-miR-NC (5  μl) were intra-articular (IA)-injected dehydrated, paraffin-embedded, and sliced into sections. F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 3 of 10 After that, H&E was used to stain the tissues. A micro- RT‑qPCR scope was used to observe pathological changes of syno- After transfection, the total RNA was extracted from vial tissues. The standard of pathological score was set as ATDC5 cells and tissues of OA rats using TRIzol rea- previous described [22]. Higher score represented greater gent (Invitrogen). Total RNA was reverse transcribed joint injury severity. into cDNA utilizing a PrimeScript product RT reagent Kit (Takara, Kyoto, Japan). Then, cDNA was amplified by polymerase chain reaction (PCR). U6 was the internal Safranin O/fast green staining reference of miR-18a-3p and GAPDH was the internal Knee joints of sham-operated control rats and OA rats reference of mRNAs. The results were analyzed using the were deparaffinized in xylene, hydrated with gradient −ΔΔCt 2 method. The primer sequences are presented in ethanol, and stained with safranin-O/fast green. Scoring Additional file 1: Table S1. criteria was introduced in this study [23]. Statistical analysis Enzyme‑linked immunosorbent assay (ELISA) The data are presented as the mean ± standard deviation For in vitro experiments, ATDC5 cells (2 × 10  cells/well) and statistics were analyzed by GraphPad Prism software were pretreated with different vectors in 6-well plates for 6.0 (La Jolla, CA, USA). Student’s t test was utilized for 2  h and then stimulated with IL-1β. For in  vivo experi- difference comparison between two groups, and one-way ments, blood samples were collected from rats infected analysis of variance followed by Tukey’s post hoc analysis with AAV. Then, serum was obtained after blood sam - were used for evaluation of differences among multiple ples were centrifuged at 6000×g for 15  min at 4  °C. The groups. A value of p < 0.05 was considered statistically concentrations of inflammatory cytokines (IL-6, IL-8 significant. and PGE2) in cells or serum were assessed using cor- responding ELISA kits (Elabscience Biotechnology, Results Wuhan, China). Absorbance values were read at 450 nm MiR‑18a‑3p overexpression improves cartilage matrix in a microplate reader (BioTek synergy HT, Bedfordshire, remodeling and inflammation in an in vitro model of OA UK). First, we used IL-1β to stimulate ATDC5 cells to con- struct an in  vitro model of OA. After IL-1β treatment, Western blotting we observed that miR-18a-3p expression in ATDC5 cells The concentration of total proteins obtained from was downregulated (Fig. 1A). miR-18a-3p expression was ATDC5 cells and tissues of OA rats was determined by increased in ATDC5 cells after transfection with miR- a BCA assay kit (Beyotime, Shanghai, China). Next, the 18a-3p mimic (Fig.  1B). Then, secretion of inflammatory proteins were loaded and separated by 8% SDS-PAGE. cytokines including IL-8, IL-6 and PGE2 was examined Then, the proteins were transferred onto PVDF mem - by ELISA. The results showed that IL-1β increased the branes and blocked with 5% skim milk for 1  h. After- release of these inflammatory cytokines in ATDC5 cells, wards, the membranes were incubated with primary and miR-18a-3p overexpression reversed IL-1β-induced antibodies including anti-MMP2 (ab92536; 1:1000), anti- increase in cytokines (Fig.  1C–E). Meanwhile, the pro- MMP3 (ab52915; 1:1000), anti-MMP9 (ab76003; 1:1000), tein levels of matrix metalloproteinases (MMP2, MMP3, anti-PDP1 (ab198261; 1:200), and anti-β-actin (ab8226; and MMP9) were increased in ATDC5 cells after IL-1β 1:1000) at 4  °C overnight and then with secondary anti- treatment compared with those in control groups and body horseradish peroxidase-labeled IgG (ab6721; were decreased in IL-1β-stimulated ATDC5 cells silenc- 1:2000) at 4 °C for 2 h. Protein bands were visualized by ing miR-18a-3p compared with those in cells transfected BeyoECL Plus (Beyotime) and analyzed using Image Lab with miR-NC (Fig.  1F). All these data suggested that 3.0 (Invitrogen). an in  vitro model of OA was successfully established. In addition, miR-18a-3p is downregulated in IL-1β- Luciferase reporter assay stimulated ATDC5 cells, and miR-18a-3p overexpression The wild-type (Wt) PDP1 3′UTR fragment contain - suppress levels of matrix metalloproteinases and proin- ing binding site of miR-18a-3p was inserted into the flammatory cytokines in the in vitro model of OA. pmirGLO vector (Promega, Madison, WI) to generate the luciferase reporter PDP1-Wt. PDP1-Mut was con- MiR‑18a‑3p targets PDP1 structed by insertion of mutated PDP1 3′UTR into the To explore the mechanism of miR-18a-3p in IL-1β- pmirGLO vector. PDP1 3′UTR Wt/Mut were transfected stimulated ATDC5 cells, potential target genes of miR- into ATDC5 cells with miR-18a-3p mimic or miR-NC 18a-3p were predicted by miRDB and the first eight genes using Lipofectamine 2000 (Invitrogen). Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 4 of 10 Fig. 1 MiR-18a-3p overexpression improves cartilage matrix remodeling and inflammation in IL-1β-stimulated ATDC5 cells. A MiR-18a-3p expression in ATDC5 cells with or without IL-1β treatment was detected by RT-qPCR. B Transfection efficiency of miR-18a-3p mimics in IL-1β-stimulated ATDC5 cells was detected by RT-qPCR. After IL-1β treatment or miR-18a-3p overexpression, C–E the secretion of inflammatory cytokines IL-8, IL-6 and PGE2 in ATDC5 cells was examined by ELISA. F Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) were examined by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001 (SNX8, FNDC5, EFNA1, ZBBX, ADCY1, PDP1, UBE2Z level in ATDC5 cells (Fig. 2E, F). Therefore, it can be con - and THSD7B) were identified (Fig.  2A). After overex- cluded that PDP1 is targeted by miR-18a-3p in ATDC5 pressing miR-18a-3p, only PDP1 was downregulated in cells. ATDC5 cells among these candidates, as shown by RT- qPCR (Fig. 2B). The binding site of miR-18a-3p on PDP1 PDP1 upregulation reserves the inhibitory effect 3′UTR was predicted by TargetScan, and the binding site of miR‑18a‑3p overexpression on levels of inflammatory is highly conserved among species (Fig.  2C). Luciferase cytokines and matrix metalloproteinases in the in vitro reporter assay was used to validate the binding relation model of OA between miR-18a-3p and PDP1 3′UTR, which revealed To identify the role of the miR-18a-3p/PDP1 regulatory that miR-18a-3p upregulation significantly decreased the axis in IL-1β-treated ATDC5 cells, rescue assays were luciferase activity of PDP1 3′UTR-Wt rather than that of performed. MiR-18a-3p overexpression downregulated PDP1 3′UTR-Mut (Fig.  2D). In addition, overexpressing PDP1 mRNA and protein levels in IL-1β-stimulated miR-18a-3p contributed to the decrease in PDP1 protein ATDC5 cells, while PDP1 upregulation reversed this F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 5 of 10 Fig. 2 MiR-18a-3p targets PDP1. A Potential target genes (SNX8, FNDC5, EFNA1, ZBBX, ADCY1, PDP1, UBE2Z and THSD7B) of miR-18a-3p were predicted by miRDB. B The expression of potential target genes in ATDC5 cells transfected with miR-18a-3p mimics or miR-NC was detected by RT-qPCR. C The binding site of miR-18a-3p for PDP1 3′UTR was predicted by TargetScan. D Luciferase reporter assay was used to validate the binding relation between miR-18a-3p and PDP1 3′UTR in ATDC5 cells. E, F PDP1 protein level in ATDC5 cells after overexpressing miR-18a-3p was examined by western blotting. **p < 0.01 trend (Fig.  3A, B). MiR-18a-3p-induced decrease in effect of miR-18a-3p overexpression on levels of matrix inflammatory cytokines (IL-8, IL-6 and PGE2) was metalloproteinases and proinflammatory cytokines in reversed by PDP1 elevation in IL-1β-stimulated ATDC5 IL-1β-stimulated ATDC5 cells. cells (Fig. 3C–E). Furthermore, miR-18a-3p upregulation led to reduction of matrix metalloproteinases (MMP2, MiR‑18a‑3p overexpression inhibits OA progression in vivo MMP3 and MMP9) in IL-1β-stimulated ATDC5 cells, To further validate the functions of miR-18a-3p in OA which was offset by PDP1 overexpression (Fig.  3F). Taken pathogenesis, in  vivo experiments were also conducted. together, PDP1 upregulation reserves the suppressive A total of 32 Wistar rats were randomly divided into Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 6 of 10 Fig. 3 PDP1 upregulation reserves the inhibitory effect of miR-18a-3p overexpression on levels of inflammatory cytokines and matrix metalloproteinases in IL-1β-stimulated ATDC5 cells. A, B PDP1 protein level in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 was examined by western blotting. C–E The secretion of inflammatory cytokines IL-8, IL-6 and PGE2 in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 was examined by ELISA. F Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) in IL-1β-stimulated ATDC5 cells after overexpressing miR-18a-3p or PDP1 were examined by western blotting. **p < 0.01, ***p < 0.001 IL-8, IL-6 and PGE2 were higher in OA model rats than four groups (n = 8/group): sham + AAV-miR-NC; in sham-operated rats, and miR-18a-3p overexpres- sham + AAV-miR-18a-3p; OA + AAV-miR-NC and sion inhibited the secretion of inflammatory cytokines OA + AAV-miR-18a-3p. According to the results from (Fig.  4C). The increase in matrix metalloproteinases H&E staining, miR-18a-3p overexpression attenuated (MMP2, MMP3 and MMP9) in OA rats was reversed the pathological changes of synovial tissues in OA rats, after upregulating miR-18a-3p (Fig.  4D, E). Finally, miR- including alleviated proliferation of synovial cells and 18a-3p expression in cartilage tissues was detected using reduced inflammatory cells (Fig.  4A). In addition, miR- RT-qPCR. After AAV-miR-18a-3p infection, miR-18a-3p 18a-3p elevation significantly reduced cartilage deg - was overexpressed in cartilage tissues of sham-operated radation of OA rats according to results of Safranin O/ rats (Fig.  4F). Compared with rats in the sham group, Fast Green Staining (Fig.  4B). Moreover, serum levels of (See figure on next page.) Fig. 4 MiR-18a-3p overexpression inhibits OA progression in vivo. A H&E staining was used to observe pathological changes of synovial tissues in the knee joint of sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p. B Safranin O-fast green/hematoxylin was used to stain histologic samples of knee joints of sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p. C Serum levels of IL-8, IL-6 and PGE2 in sham-operated control rats and OA rats infected with AAV-miR-NC or AAV-miR-18a-3p were examined by ELISA. D, E Protein levels of matrix metalloproteinases (MMP2, MMP3 and MMP9) in sham-operated control rats and OA rats treated with AAV-miR-NC or AAV-miR-18a-3p were examined by western blotting F miR-18a-3p expression in cartilage tissues of rats of four groups was detected using RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001 F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 7 of 10 Fig. 4 (See legend on previous page.) Feng et al. The Journal of Physiological Sciences (2022) 72:3 Page 8 of 10 OA model rats exhibited decreased miR-18a-3p expres- act as a protective role in OA progression; in the study sion (Fig.  4F). Additionally, AAV-miR-18a-3p success- revealing the enhancer role of miR-18a-5p in TNFα- fully upregulated miR-18a-3p in cartilage tissues of OA induced cartilage destruction and chronic inflammation model rats (Fig.  4F). Overall, miR-18a-3p expression [31], NF-κB is an inducer of miR-18a-5p and NF-κB acti- levels in vivo were consistent with results of miR-18a-3p vation is known to be involved in many chronic inflam - expression detection in IL-1β-stimulated ATDC5 cells. matory diseases [32]. In the current study, the target Overall, miR-18a-3p elevation inhibits OA progression gene PDP1 is identified to promote inflammation in in vivo. many diseases [33–35]. u Th s, we suspected that the dif - ferent role of upstream or downstream genes may affect the functions of miR-18a-3p and miR-18a-5p in arthritis. Discussion Here, we also investigated the functions of miR-18a-3p OA is a degenerative disease that poses a threat to health in ATDC5 cells after IL-1β treatment. We found that condition of humans [24]. IL-1β can induce articular IL-1β-stimulated the increase in concentration of inflam - chondrocytes to produce cytokines and chemokines, matory cytokines and matrix metalloproteinases was thus leading to inflammation [25]. IL-1β upregulates attenuated by miR-18a-3p upregulation in ATDC5 cells. MMPs to damage articular cartilage structure and unbal- More importantly, miR-18a-3p alleviated the pathologi- ance metabolism [26]. Thus, IL-1β overexpression is a cal changes of OA rats in in vivo experiments. In conclu- hallmark of OA progression [27]. In this study, IL-1β sion, miR-18a-3p inhibits OA progression by improving was used to treat ATDC5 cells to stimulate the in  vitro cartilage matrix remodeling and suppressing inflamma - pathological environment of OA. After IL-1β stimula- tion (Fig. 5). tion, the secretion of inflammatory cytokines (IL-8, IL-6 MiRNAs are gene regulators that directly bind to and PGE2) and the concentration of matrix metallopro- 3′UTR of their mRNAs to degrade transcripts or sup- teinases (MMP2, MMP3 and MMP9) were increased in press protein translation [36, 37]. In the current study, ATDC5 cells. The results suggested that an in vitro model PDP1 was identified as a target gene of miR-18a-3p and of OA was successfully established. negatively regulated by miR-18a-3p. PDP1 is also called Accumulating studies have revealed that miRNAs PDH, PDP, PDPC, PPM2A, PPM2C [38]. PDP1 overex- are involved in OA progression [16, 28, 29]. In the cur- pression leads to exhaustion of hematopoietic stem cells rent study, miR-18a-3p was downregulated in an in vitro to increase the occurrence of myeloproliferative disor- model of OA, suggesting that miR-18a-3p participated der [39]. Many studies revealed that induction of PDP1 in the pathogenesis of OA. MiR-18a-3p was reported to be downregulated in the knee anterior cruciate ligament of OA patients [20], which is consistent with our results. However, in another study, miR-18a-3p was reported to be upregulated in chondrocytes isolated from mouse and human cartilage tissues and directly target downstream gene homeobox A1 to facilitate chondrocyte apoptosis [21], which is opposite to our findings and the results of miRNA sequencing done by Bin et al. [20]. Furthermore, miR-18a, also termed miR-18a-5p, is derived from the same pri-miRNA as miR-18a-3p. MiR-18a-5p is over- expressed in primary Sjögren’s syndrome, a disease that can cause muscle and joint pain and muscle weakness [30]. MiR-18a-5p increased the expression of MMP1, inflammatory cytokines and chemoattractant proteins in rheumatoid arthritis synovial fibroblasts through NF-κB dependent manner after TNFα stimulation [31]. The pre - vious study concluded that miR-18a-5p is an enhancer of TNFα-induced cartilage destruction and chronic inflam - mation in the joint [31]. These published articles revealed Fig. 5 As shown in the diagram, IL-1β downregulates miR-18a-3p that miR-18a-3p and miR-18a-5p might play a different in ATDC5 cells. miR-18a-3p directly targets PDP1, and PDP1 role in arthritis. Moreover, the functions of a miRNA promotes the concentration of inflammatory cytokines and matrix metalloproteinase. Thus, miR-18a-3p suppresses levels of might be affected by its upstream or downstream genes. inflammatory cytokines and matrix metalloproteinase by targeting In the study revealing the miR-18a-3p/HOXA1 axis in PDP1 in IL-1β-stimulated ATDC5 cells OA [21], HOAX1 inhibits chondrocyte apoptosis and F eng et al. The Journal of Physiological Sciences (2022) 72:3 Page 9 of 10 Authors’ contributions expression is Notch-dependent, and PDP1 is involved XF and HX were the main designers of this study. XF, JL, YW and HX performed in the activation of proinflammatory macrophages [33] the experiments and analyzed the data. HX drafted the manuscript. All authors and mouse hepatic macrophages [34]. PDP1 was also read and approved the final manuscript. reported to function as an integral signaling target for Funding proinflammatory stimuli (formyl-methionyl-leucyl-phe - None. nylalanine and leukotriene B4) and anti-inflammatory Availability of data and materials mediators (lipoxins) to regulate the activation of poly- The data underlying this article will be shared on reasonable request to the morphonuclear neutrophils in neutrophil proinflam - corresponding author. matory responses [35]. Similarly, in this study, PDP1 upregulation reverses the inhibitory impact of miR- Declarations 18a-3p upregulation on inflammatory cytokines in IL-1β- Ethics approval and consent to participate stimulated ATDC5 cells. The association between PDP1 All institutional and national guidelines for the care and use of laboratory and MMPs has not been clearly reported yet. Here, we animals were followed. discovered that overexpressing PDP1 rescued the sup- Consent for publication pressive effect on protein levels of MMP2, MMP3 and Not applicable. MMP9 in IL-1β-stimulated ATDC5 cells. All these results suggested that PDP1 promotes inflammatory response in Competing interests The authors have no competing interests. OA development, and miR-18a-3p inhibits inflammation in OA by targeting PDP1. Received: 15 September 2021 Accepted: 19 January 2022 In the published article, NF-κB is an inducer of miR- 18a in TNFα-induced rheumatoid arthritis synovial fibroblasts [31]. Additionally, NF-κB activation is the principal pathway of Notch signaling pathways, and PDP1 is a target of the Notch pathway and is activated References by Notch [33, 34]. The Notch pathway exerts a proinflam - 1. 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Journal

The Journal of Physiological Sciences – Springer Journals

Published: Dec 1, 2022

Keywords: OA; miR-18a-3p; PDP1; Chondrocyte; Inflammation

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