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FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression

Arthritis Research & Therapy volume 25, Article number: 53 (2023) Cite this article

Abstract

Objectives

Increasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression.

Materials and methods

The FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2.

Results

FTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage.

Conclusions

Our results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

Introduction

Osteoarthritis (OA) is a frequent chronic irreversible joint disease characterized by degradation of cartilage extracellular matrix (ECM) and chondrocytes apoptosis, which influences an increasing number of people around the world [1, 2]. The clinical symptoms of OA patients are joint pain, joint stiffness, joint swelling, and loss of joint function, which may even lead to difficulty in daily activities [3,4,5]. Currently, non-steroidal anti-inflammatory drugs and surgical intervention are the main treatment measures for OA, but these therapeutic effects are unsatisfactory along with some side effects [6]. Therefore, it is very indispensable to superior elucidate the pathogenesis of OA and explore new therapeutic targets.

N6-methyladenosine (m6A) is a ubiquitous internal modification of eukaryotic RNA, which plays central roles in physiological and pathological processes [7]. The m6A modification has been shown to regulate different stages of RNA metabolism, including maturation, folding, export, translation, and decay, which are regulated by writers protein (METTL3, METTL14, WTAP, and etc.), erasers protein (FTO, ALKBH5 and ALKBH3), and readers protein (YTHDCs, YTHDFs, IGF2BPs and etc.) [8, 9]. Previous studies have attested that dysregulation of m6A modification and its related genes participate in the development of OA [10,11,12,13]. The earliest discovered FTO catalyzes m6A demethylation in a Fe(II)—and α-ketoglutarate-dependent manner [14]. The previous research has shown that FTO polymorphism rs8044769 alleles is a risk factor for temporomandibular joint OA, which may be involved in temporomandibular joint OA progression [15]. Nevertheless, the specific roles and potential mechanisms of FTO in OA has not been explored.

MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNAs with a length of about 22 nucleotides encoded by endogenous genes, which play important roles in joint homeostasis and the pathogenesis of OA [16]. Recently, more and more attention has been paid to the study that m6A modification regulates miRNAs maturation and then affects target gene expression [17, 18]. For examples, METTL3 accelerated the miR-221/222 maturation by mediating m6A modification, thereby resulting in the inhibition of PTEN [19]. Moreover, FTO outstandingly reduced the miR-576 formation by mediating m6A modification and then contributed to the enhancement of CDK6, thus promoting bladder cancer progression [20]. FTO significantly increased the ARL5B expression through the suppression of miR-181-3p, thus promoting the invasion and migration of breast cancer cells [21]. FTO knockdown regulated m6A-mediated pri-miR-10a processing to promote miR-10 maturation, and then inhibited MTMR3 expression, thereby promoting glioma progression [22]. These studies have disclosed that FTO plays a crucial part in multiple tumors by regulating the m6A modification of miRNAs. However, whether FTO is involved in the development of OA remains unclear.

In this study, our founding unmasked that FTO ameliorated OA cartilage damage in vitro and in vivo, whereas FTO knockdown aggravated LPS-induced chondrocytes injury in vitro. Moreover, this study revealed that FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which resulted in the increase of PRKAA2 expression, thereby inhibiting chondrocytes damage. Briefly, our founding not only revealed a novel mechanism of chondrocytes injury in OA, but also demonstrated that FTO might be a promising therapeutic target for patient with OA.

Material and methods

Human articular chondrocytes culture

As previously described [23], human articular chondrocytes were separated from articular cartilage gained from OA patient undergoing total knee replacement surgery (n = 6). Briefly, fresh articular cartilage tissues were collected and digested with 0.25% trypsin (Sigma-Aldrich). After digestion at 37 °C for 30 mim to remove fibroblasts, these articular cartilages were digested with 0.2% type II collagenase (Sigma-Aldrich) at 37 °C for 6 h and then filtered by a 100-μm cell strainer. Finally, the cells collected by centrifugation (1000 rpm/mim, 10 min) were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Gibco) in the constant temperature incubator with 5% CO2 and then observed the cell morphology under the microscope. The cells cultured to the second generation were identified as chondrocytes by immunofluorescence (Collagen II antibody) in addition to observing the cells morphology, and the chondrocytes cultured to the third generation were used for later experimental research. In addition, all clinical samples in this study were confirmed by the Ethics Committee of Zhujiang Hospital of Southern Medical University (No.2018-GJGBWK), and all participants signed written informed consent prior to the program.

Adenovirus construction and LPS stimulation

The adenovirus of FTO or PRKAA2 overexpression / knockdown and their negative control were constructed by Genechem (Nanjing, China). The adenovirus particles of miR-3591-5p mimics, miR-3591-5p inhibitor, and their negative controls were gained from Ribobio (Guangzhou, China). All cells were infected with the adenovirus on the basis of the instructions, and the infection efficiency was verified by quantitative real-time PCR (RT-qPCR) or western blotting. After 48 h of adenovirus transmission, chondrocytes were stimulated with lipopolysaccharide (LPS; Sigma-Aldrich) for the 24 h and then used for further studies. The sequences of all shRNAs, miR-3591-5p mimics, miR-3591-5p inhibitor, and their negative controls are showed in Supplementary Table 1.

RT-qPCR

Total RNAs were extracted from chondrocytes or articular cartilage by the TRIzol reagent (Takara, Japan), and then was reversed transcribed into complementary DNA by PrimeScript™ RT reagent Kit with gDNA Eraser (#RR047A, Takara). Finally, RT-qPCR amplification was performed (Roche, Indianapolis, IN, USA) on the Roche LightCycler ®96 Real-Time PCR System using BenyoFastTMSYBR Green qPCR Mix (#D7260, Beyotime). Small RNA RNU6 (U6) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as an internal reference for miR-3591-5p and other mRNAs, respectively. All genes expression levels was evaluated by 2−ΔΔCt method. All primers sequences involved in RT-qPCR were gained from Sangon Biotech (Shanghai, China) and displayed in Supplementary Table 2.

RNA m6A quantification

Total RNAs were separated from chondrocytes or articular cartilage tissues using TRIzol reagent (Takara, Japan), and then relative RNA m6A levels were quantified by m6A RNA methylation assay kit (colorimetric; Abcam, ab185912) according to the instructions. Finally, the relative m6A RNA methylation levels were computed by the following formula: m6A% = [(Sample OD-NC OD)/S] × 100%/[(PC OD-NC OD)/P], where the OD indicated absorbance value at 450 nm, NC indicated negative control, PC indicated positive control, S indicated the amount of input sample RNA, and P indicated the amount of input positive control.

Western blot

Total proteins were separated from chondrocytes and articular cartilage using pre-cooled radioimmunoprecipitation buffer (Beyotime, Haimen, China) containing protease inhibitor (Roche, Indianapolis, IN, USA). After proteins quantification and denaturation, equal quantity of proteins were isolated using electrophoresis and then transferred into a polyvinylidene difluoride membranes (Millipore, USA). Subsequently, this membrane was blocked by 5% skim milk for 1 h, and then incubated with primary antibodies overnight at 4 °C. After washing the membrane, the membrane was incubated with secondary antibody (Abcam, UK) for 2 h at room temperature. Finally, the protein band was visualized with the ECL regents (DINGGUO Biology, China) on Gel imaging system (GE Healthcare). In addition, the gray values of each band were analyzed using the Image J software. Primary antibodies in the study are as follows: FTO (ab126605, Abcam; 1:1000), ADAMTS5 (ab41037, Abcam; 1:1000), MMP13 (ab219620, Abcam; 1:1000), Collagen II (ab34712, Abcam; 1:1000), Aggrecan (ab3778, Abcam; 1:1000), PRKAA2 (ab126911, Abcam; 1:1000), and GAPDH (ab181602, Abcam; 1:2000).

Cells proliferation

The cell counting kit-8 (CCK-8) assay and a 5-ethynyl-20-deoxyuridine (EDU) assay were applied to analyze cell proliferation. For CCK-8 assay, the treated cells were dealt with Enhanced Cell Counting Kit-8 (Beyotime, #C0042) according to the instructions. The absorbance of each sample was measured by a microplate reader (Tecan, F50) at 450 nm. For EDU assay, the treated cells were conducted by EDU assay kit (Ribobio, Guangzhou, China) on the basis of the instructions. After nuclear staining using Hoechst 33,342, the images were obtained by an inverted fluorescence microscope (Mshot, MF52) and then analyzed using image J.

Cells apoptosis

Cells apoptosis were estimated via flow cytometry and Caspase-3 activity. For flow cytometry, the treated chondrocytes were performed using Annexin V-FITC/PI double-staining kit (Abcam) in the light of the instructions. The treated chondrocytes were blended with Annexin V-FITC dye and PI dye and co-incubated for 20 min at room temperature in avoid light. Finally, the apoptosis rate was detected and analyzed by BD FACSVerse™ Flow Cytometer (BD Biosciences, USA) and Cell Quest (BD Bioscience, USA) software. In addition, the caspase-3 activity was measured using the Caspase-3 Colorimetric Assay Kit (Keygen Biotech, Nanjing, China) in the light of the manufacturer’s protocol. The absorbance of each sample was detected by a microplate reader (Tecan, F50) at 450 nm.

miRNA sequencing and data analysis

The miRNA sequencing and data analysis were carried out by Shanghai Biotechnology Corporation (Shanghai, China). In short, total RNAs were separated from each sample using mirVana™ miRNA Isolation Kit (Cat #. AM1561, Austin TX, US) following the manufacturer’s standard operating procedures and then got through the electrophoresis quality inspection by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA) for subsequent detection. Subsequently, the sequencing sample library construction and cluster generation were performed. After the sequencing sample library construction and cluster generation were completed, the flow cell carrying the cluster was sequenced on the machine. The sequencing process was commanded by the data collection software provided by Illumina, and real-time data analysis was carried out. The differentially expressed miRNAs were identified with p < 0.05 and |log2fold-change (FC)|> 2.

Data sources and bioinformatics analysis

A sequence-based m6A modification site predictor (SRAMP) was used for predicting m6A modification sites on pri-miR-3591. The mRNAs expression profile related to OA progression was investigated in GSE113825 and GSE16464 datasets from Gene Expression Omnibus (GEO) database and analyzed using GEO2R. The threshold criteria for differential expressed genes were log2FC ≥  − 1.5 and p value < 0.01. The potential targets genes of miR-3591-5p were forecast through miRDB (http://mirdb.org/) and TargetScan (http://www.targetscan.org/), and then these predicted mRNAs were crossed with those mRNAs that significantly downregulated in GSE113825 and GSE16464 dataset.

RNA immunoprecipitation (RIP) analysis

The EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) was applied to execute RIP experiment. Briefly, the treated cells were lysed in RIP lysis buffer, and then these lysate was incubated with magnetic beads coupled with the anti-Ago2 antibody (ab186733, Abcam), anti-DGCR8 antibody (ab191875, Abcam), anti-m6A antibody (ab286164; Abcam), or Immunoglobulin G antibody (IgG, ab6715, Abcam) at 4 °C for 6 h. After washing, co-precipitated RNAs were extracted from magnetic beads complex using TRIzol reagent (Takara, Japan) and then RT-qPCR was performed using specific primers.

In vitro pri-miRNA processing assays

In vitro pri-miR-3591 processing assays was carried out according to the methods previously reported [24, 25]. In brief, the mutant pri-miR-3591[m6A] were generated by replacing adenosine with thymine, and then pri-miR-3591[m6A]-WT or pri-miR-3591[m6A]-Mut and pri-miR-1–1 (control) were incubated with whole cellular lysates of chondrocytes co-overexpressing DROSHA and DGCR8. Finally, total RNAs extracted from reaction products was subjected to RT-qPCR analysis.

Luciferase reporter assay

To further validate the effect of m6A modification on miR-3591-5p expression, m6A modification sites on pri-miR-3591 sequences were predicted through SRAMP. We identified one putative m6A recognition sites on pri-miR-3591 sequences. According to this predicted results, mutagenesis from adenosine to thymine was generated by QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA) in the light of the instruction, and then the wild-type and mutant pri-miR-3591 reporter vectors were co-transfected with shFTO plasmid into chondrocytes. Transfer after 48 h, luciferase activities were assessed by a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) in accordance with the instructions. For the verification of miR-3591-5p and PRKAA2 targeted interactions, the wild-type or mutant-type of PRKAA2 (containing the binding site of miR-3591-5p) was cloned into the luciferase vector, and then transfected into in chondrocytes together with miR-3591-5p mimics or NC mimics, respectively. Transfer after 48 h, luciferase activities were assessed by a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) on the base of the instructions. Finally, the signal value of Renilla luciferase was normalized as an internal reference.

Destabilizing the medial meniscus (DMM)-induced OA mouse model

All animal experiments were executed in the light of the Guide for the Care and Use of Laboratory Animals and confirmed by the Animal Care Committee of Zhujiang Hospital of Southern Medical University (No: 2018-GJGBWK). The male C57BL/6 mice (SPF, 8-week-old) were randomly divided into four groups: sham (n = 6), model (n = 6), model + adeno-associated virus-negative control (AVV-NC) (n = 6), and model + AVV-FTO groups (n = 6). After acclimating for one week, mice in other groups were treated with DMM surgery to induce OA model as previously described except for the sham groups [26]. Mice in sham groups was underwent only the skin of the right knee joint incision. The AAV-NC and AAV-FTO were constructed by HANBIO (Shanghai, China). The mice in model + AAV-NC and model + AAV-FTO groups were intra-articularly injected with 10 μL of AAV-NC and AAV-FTO (1 × 1013 vg/ml) through the medial parapatellar area at 2 weeks after the DMM operation, respectively. At the same point in time, the mice in the sham and model groups were intra-articularly injected with the equal volume of normal saline. After 8 weeks post-surgery, knee joints were harvested from euthanized mice for later histological analysis and molecular analysis.

Histological analysis

The fresh knee joints were fixed with 4% paraformaldehyde, decalcified in 15% EDTA-2Na, dehydrated in gradient ethanol, embedded in paraffin, and then sliced into 5-μm sections. After dewaxing in xylene and hydration with a graded ethanol series, the sections were stained with safranin-O/fast green (Yeasen) and hematoxylin and eosin (HE, Sigma-Aldrich), respectively. The images of each section were visualized and photographed under an inverted fluorescence microscope (Mshot, MF52). In addition, the degree of articular cartilage damage in OA mice was assessed by Osteoarthritis Research Society International (OARSI) score as described previously [27].

Statistical analysis

All data in present research are indicated as the mean ± standard deviation (SD). The GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) was applied to perform statistical analysis and draw diagram. Differences between groups were analyzed using Student’s t-test or two-way ANOVA. The correlation between FTO mRNA expression and total m6A levels was evaluated by Pearson correlation coefficient. A p-value < 0.05 was considered statistically significant and each experiment was carried out in triplicate.

Results

Downregulation of FTO expression in OA cartilage and LPS-stimulated chondrocytes

To confirm the expression of FTO in cartilage tissues of OA, the expression of FTO were detected by RT-qPCR in cartilage tissues of mice with or without OA. RT-qPCR analysis revealed that the FTO was significantly downregulated in the cartilage tissues of the mice with OA compared with the mice without OA (Fig. 1A). Then, we used RNA m6A colorimetry to detect the total m6A levels in RNA extracted from cartilage tissues of mice with or without OA, and correlation analysis indicated that FTO mRNA expression was markedly negative correlated with the total m6A levels in OA cartilage (Fig. 1B). Subsequently, we also studied the changes of FTO expression and m6A levels under the LPS concentration gradient in the OA cell model and found that the expression of FTO significantly decreased with the increase of LPS concentration in a concentration dependent manner (Fig. 1C), whereas the m6A levels significantly enhanced with the LPS concentration increasing (Fig. 1D). Further overexpression of FTO in LSP-induced OA chondrocytes was confirmed by western blotting and RT-qPCR (Fig. 1E) and found that the FTO overexpression significantly suppressed the total m6A levels induced by LPS (Fig. 1F). Taken together, our findings showed that FTO was outstandingly downregulated in OA cartilage tissues and LPS-stimulated chondrocytes, and the FTO expression was significantly negative correlated with the total m6A levels.

Fig. 1
figure 1

Downregulation of FTO in OA and LPS-stimulated chondrocytes. A RT-qPCR results attested that the mRNA expression of FTO was significantly downregulated in the cartilage tissues of OA mice. B Pearson correlation coefficient analysis certified that the mRNA expression of FTO was markedly negatively related with the total m6A levels in cartilage tissue of OA mice (R =  − 0.89). C After the chondrocytes were dealt with different concentration gradients of LPS, the mRNA and protein expression of FTO was evaluated by RT-qPCR and western blotting. D After the chondrocytes were dealt with different concentration gradients of LPS, the relative m6A levels were detected by the m6A RNA methylation assay kit. E RT-qPCR and western blot were applied to confirm the overexpression of FTO in LPS-treated chondrocytes. F The m6A levels were detected using m.6A RNA methylation assay kit in different groups. Sham, mice without DMM; model, DMM surgery-induced OA mice; LPS, lipopolysaccharide; FTO, FTO overexpression adenovirus; mock, negative control corresponding to FTO overexpression adenovirus; n = 3 ~ 6, **p < 0.01, ***p < 0.001

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Effects of FTO on LPS-induced chondrocyte damage in vitro

To explore the roles of FTO in chondrocytes, the chondrocytes with or without overexpressing FTO were treated with or without LPS for 24 h. CCK-8 results indicated that LPS treatment remarkably reduced cell viability, while FTO overexpression reversed this process (Fig. 2A). Consistently, EDU experiments also showed that FTO overexpression significantly reversed the inhibition of LPS on chondrocyte proliferation (Fig. 2B and D). In addition, flow cytometry data indicated that FTO overexpression significantly impaired the elevated levels of apoptosis induced by LPS treatment (Fig. 2C and E). Consistent with flow cytometry, we found that upregulation of FTO dramatically reduced the LPS-induced caspase-3 activity in chondrocyte (Fig. 2F). Since the degradation of articular cartilage is the main clinical symptoms of OA, western blot was used for detecting the ECM degradation-related protein expression. The results attested that FTO overexpression inhibited the expression of major cartilage-degrading metalloproteinase (MMP-13 and ADAMTS-5) and enhanced the expression of chondrogenic markers (Collagen II and Aggrecan) in LPS-treated chondrocytes (Fig. 2G). In addition, the FTO knockdown was confirmed in LSP-induced OA chondrocytes using western blotting and RT-qPCR (Supplementary Fig. 1A). The results of CCK-8 and EDU assay attested that downregulation of FTO significantly aggravated the inhibition of LPS on chondrocyte proliferation (Supplementary Fig. 1B and C). Next, we assessed the effects of FTO knockdown on chondrocyte apoptosis. Flow cytometric analysis revealed that the cell apoptosis in LPS-induced chondrocytes was markedly increased following FTO downregulation (Supplementary Fig. 1D and F). And consistent with the trend of flow cytometry, the results of caspase-3 activity detection indicated that downregulation of FTO notably promoted the LPS-induced caspase-3 activity in chondrocytes (Supplementary Fig. 1E). In addition, the results of western blotting attested that FTO knockdown outstandingly enhanced the major cartilage-degrading metalloproteinase (MMP-13 and ADAMTS-5) protein expression and repressed the protein expression of chondrogenic markers (Collagen II and Aggrecan) in LPS-treated chondrocytes (Supplementary Fig. 1G), suggesting that knockdown of FTO significantly inhibited the chondrogenic capacity of chondrocytes. Taken together, our data suggested that overexpressing FTO promoted proliferation, suppressed apoptosis, and ameliorated ECM degradation in LPS-induced chondrocyte, whereas knockdown of FTO suppressed the proliferation, promoted the apoptosis, and aggravated ECM degradation in LPS-induced chondrocytes.

Fig. 2
figure 2

FTO overexpression aggravated alleviated LPS-induced chondrocyte damage in vitro. Chondrocytes were transfected with FTO or mock and then treated with 40 ng/mL LPS for 24 h. A CCK-8 was applied to evaluate the viability of chondrocytes. B and D EDU staining was applied to measure the proliferation ability of chondrocytes. C and E The apoptosis of chondrocytes was evaluated by flow cytometry. F The caspase-3 activity was evaluated by caspase-3 activity kit. G Western blotting was applied to assess the expression of MMP13, ADAMTS5, Aggrecan, and COL2A1. LPS, lipopolysaccharide; FTO, FTO overexpression adenovirus; mock, negative control corresponding to FTO overexpression adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

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FTO regulated the miR-3591-5p maturation in an m6A-dependent manner base on miRNA-Seq analysis

To investigate the molecular mechanism of FTO in cartilage injury of OA, we identified miRNAs regulated by FTO using miRNA-sequencing analysis. We found that two significantly downregulated miRNAs (hsa-miR-3591-5p and hsa-miR-218-5p) and three markedly upregulated miRNAs (hsa-miR-1185–2-3p, hsa-miR-3679-5p, and novel.39) in LPS-treated chondrocytes were reversed by FTO overexpression (Fig. 3A). Since the decreased m6A levels leads to the arrest of pri-miRNA processing, we selected miRNAs (hsa-miR-3591-5p and hsa-miR-218-5p) negatively associated with FTO and predicted m6A modification sites on pri-miR-3591 and pri-miR-218 RNA sequences using SRAMP. The results showed that an AGACU m6A sequence motif in RNA sequences of pri-miR-3591 (Fig. 3B), whereas the site was not found in pri-miR-218. So miR-3591-5p was selected for further study. Further RT-qPCR analysis confirmed that pri-miRNA-3591 was significantly accumulated in FTO-overexpressing chondrocytes and remarkably reduced in FTO-inhibiting chondrocytes, while opposite results were obtained for the expression of pre-miR-3591 and miR-3591-5p (Fig. 3C), suggesting that FTO might regulate the miR-3591-5p maturation. To further investigate the effect of m6A modification on pri-miR-3591 processing, the MeRIP coupled with RT-qPCR was used to detect the enrichment level of pri-miR-3591 in FTO overexpression or knockdown chondrocyte and found that overexpression of FTO dramatically decreased pri-miR-3591 m6A levels, while knockdown of FTO markedly increased pri-miR-3591 m6A levels (Fig. 3D), while the pri-miR-3591 wild type dual-luciferase reporter (containing potential m6A sites, pri-miR-3591 [m6A]-WT) and pri-miR-3591 mutant type dual-luciferase reporter (the adenosine base in m6A consensus sequences replaced with thymine to abolish the m6A modification, pri-miR-3591 [m6A]-Mut) was transfected into chondrocytes with or without FTO knockdown for the dual-luciferase reporter assay, and the results revealed that the luciferase activity of the wild-type pri-miR-3591[m6A] was significantly increased by FTO knockdown, but the luciferase activity of the mutant-type pri-miR-3591[m6A] did not effect by FTO knockdown, indicating the pri-miR-3591 processing was modulated by FTO-associated m6A modification (Fig. 3E). To further confirm a direct role of m6A in miR-3591-5p maturation, we carried out RIP with DGCR8 antibody and in vitro RNA processing assays. The RIP with DGCR8 antibody followed by RT-qPCR demonstrated that the DGCR8-bound pri-miR-3591 expression remarkably decreased in chondrocytes with FTO overexpression compared with that in chondrocytes without FTO overexpression (Fig. 3F). We further executed in vitro RNA processing assays using in vitro transcribed pri-miR-3591[m6A]-WT or pri-miR-3591 [m6A]-Mut and whole cell lysates of chondrocytes with DGCR8 and DROSHA overexpression. RT-qPCR analysis indicated that the processing rate of pri-miR-3951 to pre-miR-3591 and miR-3591-5p in pri-miR-3591[m6A]-Mut was outstandingly decreased compared with that of in pri-miR-3591[m6A]-WT (Fig. 3G). Collectively, the above results demonstrated that FTO regulated the miR-3591-5p maturation in an m6A-dependent manner.

Fig. 3
figure 3

FTO regulated the miR-3591-5p maturation in an m6A-dependent manner base on miRNA-Seq analysis. A Heatmap showed miRNAs expression profile between the mock group, LPS + mock group and LPS + FTO group by miRNA-seq analysis. B The sequences of pre-miR-3591, miR-3591-5p, and the potential m6A motif (AGACU) splicing site were highlighted with different colors. C Chondrocytes were transfected with shFTO or shNC or mock or FTO, and then dealt with 40 ng/mL LPS for 24 h. RT-qPCR was used to assess the mRNA expression pri-miR-3591, pre-miR-3591, and miR-3591-5p. D Chondrocytes were transfected with shFTO or shNC or mock or FTO, and then, the pri-miR-3591 [m6A] levels was evaluated using MeRIP coupled with RT-qPCR analysis. E Relative luciferase activity of the wild-type and mutant-type pri-miR-3591 reporter vectors were assessed in FTO knockdown chondrocytes. F RIP combined with RT-qPCR was applied to analyze the pri-miR-3591 binding to DGCR8 in chondrocytes with or without overexpressing FTO. G Quantification of miR-3591-5p, pre-miR-3591, and pri-miR-3591 in the reaction mixture was detected by RT-qPCR, and the results indicated that mutation of [m.6A] pri-miR-3591 abolished pri-miR-3591 processing in the in vitro reaction system. LPS, lipopolysaccharide; FTO, FTO overexpression adenovirus; mock, negative control corresponding to FTO overexpression adenovirus; shFTO, FTO knockdown adenovirus; shNC, negative control corresponding to shFTO adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

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miR-3591-5p aggravated LPS-induced chondrocytes damage in vitro by targeting regulation of PRKAA2

To investigative the regulatory mechanism of miR-3591-5p in OA, the common target mRNAs of miR-3591-5p predicted by TargetScan and miRBD were intersected with the significantly downregulated mRNA in OA cartilage from GSE113825 and GSE16464 analysis, uncovering that PRKAA2 might be a potential target gene of miR-3591-5p (Fig. 4A). After treatment of cells with mimic or inhibitor of miR-3591-5p, the results of RT-qPCR unmasked that overexpressing miR-3591-5p markedly downregulated PRKAA2 expression, whereas inhibition of miR-3591-5p outstandingly upregulated PRKAA2 expression, suggesting that PRKAA2 was negatively regulated by miR-3591-5p (Fig. 4B). Then, the interaction between PRKAA2 and miR-3591-5p was confirmed by RIP and a dual-luciferase reporter assay. The RIP with Ago2 antibody followed by RT-qPCR uncovered that the expression of PRKAA2 and miR-3591-5p were significantly increased in the miRNA complex (Fig. 4C). In addition, the prediction results showed that PRKAA2 had two potential binding sites with miR-3591-5p, and the dual-luciferase reporter assay revealed that the luciferase activity of PRKAA2-WT#1 was significantly decreased by miR-3591-5p overexpression, but the luciferase activity of PRKAA2-Mut#1, PRKAA2-WT#2, and PRKAA2-Mut#2 did not effect by the overexpression of miR-3591-5p (Fig. 4D), suggesting that the binding site#1 was the true miR-3591-5p targeting site. In addition, the miR-3591-5p inhibition significantly enhanced the mRNA and protein expression PRKAA2 in LPS-treated chondrocytes (Fig. 4E and H), while the miR-3591-5p overexpression markedly decreased the PRKAA2 expression in LPS-treated chondrocytes (Fig. S2A and S2D). All together, the above data showed that PRKAA2 was a direct downstream target genes of miR-3591-5p in chondrocytes.

Fig. 4
figure 4

miR-3591-5p aggravated LPS-induced chondrocytes damage in vitro by targeting regulation of PRKAA2. A Common target genes predicted by TargetScan and miRBD were intersected with the significantly downregulated mRNA in OA cartilage from GSE113825 and GSE16464 analysis. B The expression levels of miR-3591-5p and PRKAA2 were tested by RT-qPCR in chondrocytes with miR-3591-5p mimics or inhibitor. C RIP-Ago2 analysis indicated that interaction between miR-3591-5p and PRKAA2. D Interaction between miR-3591-5p and PRKAA2 was affirmed by the dual-luciferase reporter assay. E Chondrocytes were transfected with miR-3951 inhibitor or NC inhibitor, and then treated with 40 ng/mL LPS for 24 h. RT-qPCR was used to detect the mRNA expression miR-3591-5p and PRKAA2. F Chondrocytes were transfected with miR-3951 inhibitor or a combination of miR-3951 inhibitor and shPRKAA2 and then treated with 40 ng/mL LPS for 24 h. CCK-8 was used to detect the viability of chondrocytes. G Chondrocytes were transfected with miR-3951 inhibitor or a combination of miR-3951 inhibitor and shPRKAA2 and then treated with 40 ng/mL LPS for 24 h. The apoptosis of chondrocytes was tested by flow cytometry. H Chondrocytes were transfected with miR-3951 inhibitor or a combination of miR-3951 inhibitor and shPRKAA2 and then treated with 40 ng/mL LPS for 24 h. Western blotting was applied to assess the expression of MMP13, ADAMTS5, Aggrecan, and COL2A1. LPS, lipopolysaccharide; NC mim., negative control corresponding to miR-3591-5p mimics adenovirus; miR mim., miR-3591-5p mimics adenovirus; NC inh., negative control corresponding to miR-3591-5p inhibitor adenovirus; miR inh., miR-3591-5p inhibitor adenovirus; shPRKAA2, PRKAA2 knockdown adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

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To further investigate the miR-3591-5p/PRKAA2 axis in OA chondrocytes biological function, we transfected miR-3591-5p inhibitor along or together with shPRKAA2 into chondrocytes treated with LPS and executed a series of rescue experiments. Our results certified that downregulation of miR-3591-5p promoted proliferation, suppressed apoptosis, and suppressed ECM degradation in LPS-treated chondrocyte, and these phenomena were reversed by the inhibition of PRKAA2 (Fig. 4F, G, and H). Moreover, our data indicated that overexpression of miR-3591-5p inhibited proliferation, promoted apoptosis, and aggravated ECM degradation in LPS-treated chondrocytes, but the above effects were weakened by PRKAA2 overexpression (Supplementary Fig. 2B, C, and D). Taken together, these results demonstrated that miR-3591-5p aggravated cartilage injury of OA by sponging PRKAA2.

FTO inhibited LPS-induced chondrocyte damage through regulating miR-3591-5p/PRKAA2 axis

To further confirm whether FTO regulated PRKAA2 expression by inhibiting miR-3591-5p expression, RT-qPCR and western blot disclosed that FTO overexpression or miR-3591-5p overexpression resulted in significantly increasing or decreasing PRKAA2 expression in LPS-treated chondrocytes, but the above effect was partially weakened by co-transfection of overexpressed FTO and miR-3591-5p (Fig. 5A and B), suggesting that FTO increased PRKAA2 expression by inhibiting miR-3591-5p. To further confirm FTO/miR-3591-5p axis regulated chondrocyte biological function by executing a series of rescue experiments, the results of caspase-3 activity assay and flow cytometry assay showed that miR-3591-5p overexpression could partially reverse the decreased apoptosis in LPS-treated chondrocytes mediated by FTO overexpression (Fig. 5C and D). The results of CCK-8 and EDU assay showed that miR-3591-5p overexpression could partially reverse the increased proliferation in LPS-treated chondrocytes mediated by FTO overexpression (Fig. 5E and F). The results of western blot indicated that miR-3591-5p overexpression could partially reverse the reduced ECM degradation in LPS-treated chondrocytes mediated by FTO overexpression (Fig. 5G). Next, we verified FTO inhibited LPS-induced chondrocytes damage through regulating PRKAA2. The results of CCK-8 and EDU disclosed that PRKAA2 knockdown or FTO overexpression significantly decreased or increased the cell proliferation in chondrocytes with LPS, whereas the above effects were reversed by co-transfection of shPRKAA2 or FTO (Fig. 6A and B). Flow cytometry assay and caspase-3 activity assays showed that PRKAA2 knockdown or FTO overexpression significantly enhanced or reduced the apoptosis in chondrocytes with LPS, whereas the above effects were reversed by co-transfection of shPRKAA2 and FTO (Fig. 6C, D, and E). Western blotting showed that PRKAA2 knockdown or FTO overexpression significantly aggravated or restrained the ECM degradation in chondrocytes with LPS, as manifested by increased or inhibited expression of ADAMTS5 and MMP13, and decreased or enhanced expression of Collagen II and Aggrecan, whereas the above effects were reversed by co-transfection of shPRKAA2 or FTO (Fig. 6F). Taken together, our data demonstrated that FTO inhibited LPS-induced chondrocyte damage through regulating miR-3591-5p/PRKAA2 axis.

Fig. 5
figure 5

FTO inhibited LPS-induced chondrocyte damage through downregulating miR-3591-5p. Chondrocytes were transfected with FTO overexpression adenovirus or a combination of FTO overexpression adenovirus and miR-3951 mimics and then treated with 40 ng/mL LPS for 24 h. A The expression of miR-3591-5p was assessed by RT-qPCR. B The mRNA and protein expression of PRKAA2 was measured by both RT-qPCR and western blotting. C The caspase-3 activity was evaluated by caspase-3 activity kit. D The apoptosis of chondrocytes was evaluated by flow cytometry. E CCK-8 was detected the viability of chondrocytes. F EDU staining was used to assess the proliferation ability of chondrocytes. G Western blotting was applied to detect the expression of MMP13, ADAMTS5, Aggrecan, and COL2A1. LPS, lipopolysaccharide; miR mim., miR-3591-5p mimics adenovirus; FTO, FTO overexpression adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

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Fig. 6
figure 6

FTO inhibited LPS-induced chondrocyte damage through upregulating PRKAA2. Chondrocytes were transfected with FTO overexpression or shPRKAA2 or a combination of FTO overexpression and shPRKAA2 and then treated with 40 ng/mL LPS for 24 h. A CCK-8 was used to detect the viability of chondrocytes. B EDU staining was applied to detect the proliferation ability of chondrocytes. D and F The apoptosis of chondrocytes was assessed by flow cytometry. E The caspase-3 activity was measured by caspase-3 activity kit. F Western Blot was applied to assess the protein expression of MMP13, ADAMTS5, Aggrecan, and COL2A1. LPS, lipopolysaccharide; shPRKAA2, PRKAA2 knockdown adenovirus; FTO, FTO overexpression adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

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FTO ameliorates the progression of OA in mice

To demonstrate FTO affects the progression of OA in vivo, the DMM-induced OA mice were injected intra-articularly with AAV-FTO or AAV-NC. The results of western blotting disclosed that the FTO was outstandingly downregulated in articular cartilage of OA mice compared with that of in articular cartilage of mice without OA, while AAV-FTO treatment significantly enhanced FTO expression in articular cartilage of OA mice (Fig. 7A). Then, western blotting was applied to estimate the protein expression of cartilage degeneration markers (MMP-13 and ADAMTS-5) and the chondrogenic markers (Collagen II and Aggrecan) in articular cartilage, and the results showed that overexpression of FTO obviously suppressed the expression of cartilage degeneration markers (MMP-13 and ADAMTS-5) and outstandingly augmented the expression of chondrogenic markers (Collagen II and Aggrecan) in articular cartilage of OA mice (Fig. 7A). Meanwhile, the results of RT-qPCR indicated that overexpression of FTO significantly inhibited the miR-3591-5p expression in articular cartilage of OA mice, whereas markedly enhanced the expression of PRKAA2 (Fig. 7B). Finally, we used the HE and Safranin O-fast green to investigate cartilage surface histopathological changes after intra-articular injection in OA mice with AAV-FTO. The results showed that OA mice showed more severe degenerative OA changes than the sham mice; these modifications were obviously ameliorated by overexpression of FTO (Fig. 7C). Consistent with histological analyses, OARSI scores in articular cartilage of OA mice prominently increased compared with that in articular cartilage of mice without OA, but the increased OARSI scores were obviously inhibited following overexpression of FTO (Fig. 7D). Taken together, our data suggested that overexpression of FTO alleviated OA progression and regulated miR-3591-5p/PRKAA2 axis in OA mice.

Fig. 7
figure 7

FTO ameliorated the OA progression in mice. A The protein expression of FTO, MMP13, ADAMTS5, Aggrecan, and COL2A1 was evaluated by western blotting in the knee cartilage of OA mice with or without AAV-FTO treatment. B RT-qPCR was applied to assess the expression of miR-3591-5p in the knee cartilage of OA mice with or without AAV-FTO treatment. C The section of the knee joints was stained by HE staining and Safranin O & Fast Green staining, respectively. D OARSI scores of the knee joints in OA mice with or without AAV-FTO treatment. Sham, mice without DMM; model, DMM surgery-induced OA mice; AAV-NC, negative control corresponding to FTO overexpression adeno-associated virus; AAV-FTO, FTO overexpression adeno-associated virus; n = 6, *p < 0.05, **p < 0.01, ***p < 0.001

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Discussion

The critical roles of m6A are increasingly important in recent years [28, 29]. The m6A modification can regulate a range of biological progression, such as maturation, alternative splicing, and mRNA translation [30]. As a ubiquitous epitranscriptome modification in most eukaryotic mRNAs, catalytic enzymes affecting m6A levels have been widely introduced, including enzymes (METTL3, METTL14, and WTAP) that promote increased m6A levels and enzymes (FTO and ALKBH5) that wipe off m6A from RNA [31,32,33,34]. There are few studies on their role and regulatory mechanism in OA. Liu et al. verified that METTL3 promoted the occurrence and development of experimental OA by mediating the inflammation and apoptosis of chondrocyte [35]. A latest research showed that METTL3 regulated autophagy-GATA4 axis to intensify cellular senescence and OA progression by mediating m6A modification of ATG7 [10]. However, other essential m6A regulatory enzymes have not been reported in OA yet.

Chondrocytes, as a single cell type in cartilage tissue, have a central role in maintaining cartilage damage and remodeling [36, 37]. Moreover, chondrocyte apoptosis and ECM degradation are intently related to the occurrence and development of OA [38, 39]. Hence, suppression of chondrocyte apoptosis and ECM degradation are considered to be a therapeutic target for OA. In this study, our results established that upregulation of FTO inhibited chondrocyte apoptosis and ECM degradation in vivo and in vitro, uncovering that FTO is a promising therapeutic target molecule for OA.

It has been reported that in addition to protein-coding genes, large non-coding RNAs function as gene regulators during the OA progression [40, 41]. MiRNA processing is also regulated specifically for OA. However, there are no reports exploring the biological function of m6A modification during miRNA processing in OA. In this study, our data attested that FTO negatively regulated the expression of miR-3591-5p and positively regulated pri-miR-3591. At the same time, we analyzed the pri-miR-3591 sequence and found that it also contained the “RRACH” motif which has been confirmed as a highly conserved common sequence for RNA m6A modification [42, 43]. Further molecular experiments revealed that knockdown of FTO mediated the m6A modification of pri-miR-3591 to promote the maturation of miR-3591-5p in OA. Previous studies have identified miR-3591-5p was markedly increased in the serum of patients with glioblastoma [44] and patients with chronic HBV infection [45]. Lu et al. demonstrated that miR-3591-5p is molecule required for radiation-induced TGF-β activation [46]. However, the biological functions of miR-3591-5p in OA have not been reported. Our founding demonstrated that miR-3591-5p aggravated LPS-induced chondrocytes damages, whereas miR-3591-5p knockdown obviously inhibited LPS-induced chondrocytes damages. These results implied that miR-3591-5p downregulated by FTO with m6A modification played a protective role in OA progression.

Current studies have disclosed that PRKAA2 (also known as AMPK) plays a central role in regulating joint homeostasis and OA development, and targeting PRKAA2 is expected to become a therapeutic strategy for OA [47]. PRKAA2 knockdown in chondrocytes aggravated chondrocytes apoptosis, degradation of cartilage ECM, and inflammatory response, leading to disruption of joint homeostasis and exacerbation of OA progression [48], but PRKAA2 activity outstandingly ameliorated the severity of knee joint cartilage destruction in mice with OA[49,50,51]. However, the relationship between miR-3591-5p and PRKAA2 has not been explored yet. Our results uncovered that miR-3591-5p significantly suppressed PRKAA2 expression by sponging PRKAA2 in OA chondrocytes. Furthermore, our data also certified that overexpression of PRKAA2 obviously reduced the damage of miR-3591-5p on OA chondrocytes, while knockdown of PRKAA2 significantly lessened the protective effect of miR-3591-5p on OA chondrocytes. Interestingly, our findings also uncovered that FTO inhibited the LPS-induced chondrocyte damage by regulating miR-3591-5p/PRKAA2 axis in vitro.

In conclusion, our data revealed that FTO alleviated OA cartilage damage by the FTO/miR-3591-5p/PRKAA2 axis, which offered fire-new insights into the pathogenesis and therapeutic strategies for OA.

Availability of data and materials

All data are presented in this paper.

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Funding

This study was funded by Science and Technology Planning Project of Guangzhou City of China (Grant Number: 202002030204), Medical Scientific Research Foundation of Guangdong Province of China (Grant Number: A2021463), Medical Scientific Research Foundation of Guangdong Province of China (Grant Number: A2020071), and Natural Science Foundation of Guangdong Province of China (Grant Number: 2021A1515011545).

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Author notes

  1. Wengang Liu and Tao Jiang contributed equally to to this work.

Authors and Affiliations

  1. Department of Orthopedics, Guangdong Provincial Second Hospital of Traditional Chinese Medicine, Guangzhou, 510095, China

    Wengang Liu, Tao Jiang, Anan Li & Chao Lu

  2. The Fifth Clinical College of Guangzhou, University of Chinese Medicine, Guangzhou, 510405, China

    Wei Zheng & Jiayuan Zhang

  3. Orthopedics Center, Zhujiang Hospital of Southern Medical University, Guangzhou, 510000, China

    Zhaowei Lin

  4. Department of Joint and Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou, 510000, China

    Zhaowei Lin

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  1. Wengang Liu
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Contributions

Wengang Liu and Zhaowei Lin carried out experimental design and data analysis and interpretation. Tao Jiang, Wei Zheng, Jiayuan Zhang, Anan Li, and Chao Lu collected clinical specimens and data collection. Wengang Liu, Tao Jiang, and Zhaowei Lin wrote and completed the manuscript. All authors reviewed and approved the submitted version of the manuscript.

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Correspondence to Zhaowei Lin.

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Supplementary Information

Additional file 1: Supplementary Figure 1.

FTO knockdown aggravated LPS-induced chondrocyte damage in vitro. Supplementary Figure 2. miR-3591-5p overexpression aggravated LPS-induced chondrocytes damage by inhibiting PRKAA2. Supplementary Table 1. The sequences of shRNAs, miR-3591-5p inhibitor/mimics, and their negative control in this study. Supplementary Table 2. Primers used for RT-qPCR and other assays in this study.

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Liu, W., Jiang, T., Zheng, W. et al. FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression. Arthritis Res Ther 25, 53 (2023). https://doi.org/10.1186/s13075-023-03035-5

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Arthritis Research & Therapy

ISSN: 1478-6362

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