FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs

FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs Daohong Zhao, Bo Zhao, Jia Zhong, Hong Chen, Jun Zhang, Chaoran Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4334272/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Circular RNAs (circRNAs) are a class of noncoding RNAs that are involved in chondrogenic differentiation, and N6-methyladenosine (m 6 A) broadly exists in circRNAs. Materials and methods A joint injury model was constructed on Diannan small-ear (DSE) pigs. Transfections were constructed using Lipofectamine 2000. Real-time quantitative PCR (qPCR), Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR), and western blotting analyses were performed. Alcian blue staining tested the chondrogenic differentiation ability. The potential m 6 A methylation modification enzymes and sites of circZCCHC14 were predicted in m6Avar and SRAMP databases. RNA pull-down and RIP assays were conducted to determine the interaction between circZCCHC14 and FTO. Results CircZCCHC14 expression and the m 6 A methylation level were increased in joint injury DSE pigs. m 6 A methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation. FTO was decreased, circZCCHC14 and m 6 A methylation level were increased under inflammatory conditions. FTO is one essential m 6 A demethylase enzyme of circZCCHC14. m 6 A demethylase enzyme FTO regulated the expression levels of circZCCHC14. m 6 A demethylase enzyme FTO positively regulated the expression of miR-181a. FTO m 6 A demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Conclusion Our data showed the physiological significance of FTO m 6 A demethylase in regulating axis circZCCHC14/miR-181a/GREM1 and BMP2, providing a potentially effective therapeutic target for the treatment of joint injury or Osteoarthritis (OA). circZCCHC14 m6A methylation chondrogenic differentiation Bone marrow mesenchymal stem cells FTO Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Osteoarthritis (OA) is one of the joint function defect diseases, with no effective clinical therapies ( 1 ). The treatment of articular cartilage defects has always been a major challenge that joint surgeons urgently need to solve. Clinical treatment methods consist of drug-conservative treatment, joint cleaning, grinding and shaping surgery, microfracture surgery, and autologous cartilage transplantation, all of which showed limited therapeutic effects and cannot repair articular cartilage defects completely ( 2 ). In recent years, the development of tissue engineering technology has opened up new avenues for the treatment of cartilage defects ( 3 , 4 ). Seed cells, scaffold materials, and growth-inducing factors were three key factors of tissue engineering technology and have been a hot study topic in recent years. Mesenchymal stem cells (MSCs) have become ideal seed cells for cartilage tissue engineering due to their multidirectional differentiation ability, easy cultivation, and expansion characteristics ( 5 , 6 ), among which bone marrow mesenchymal stem cells (BMSCs) were commonly used ( 7 ). How to induce and regulate the directional differentiation of MSCs into chondrocytes is currently an important research issue in cartilage tissue engineering. Currently, the relevant regulatory mechanisms of MSCs chondrogenic differentiation are not fully understood. Therefore, exploring the relevant regulatory mechanisms of MSCs chondrogenic differentiation may provide support for exploring more effective differentiation-promoting strategies. Circular RNA (circRNA), mainly located in the cytoplasm and exosomes, is not affected by RNA external enzymes, thus showing more stable and conserved features than other ncRNAs, which have been identified as potential noninvasive biomarkers and as drugs for the OA treatment ( 8 ). In recent years, some studies have confirmed that circRNAs have important functions in the maintenance and differentiation of stem cells ( 9 – 11 ). It has also been recognized in studying the growth and differentiation of cells, the directional development of tissue organs, and the determination of disease treatment targets ( 12 ). As reported, circRNAs have played an important regulatory role in bone and cartilage differentiation ( 8 ). For example, circCREBBP actives the Smad1/5 pathway through the transforming growth factor beta 2 (TGFβ2)/activin receptor-like kinases 1(ALK1) axis, thus modulating cartilage degradation ( 13 ). Our previous study revealed the role of circZCCHC14 in affecting the chondrogenic differentiation ability of peripheral blood‑derived mesenchymal stem cells (PBMSCs) through the miR-181a/Gremlin 1(GREM1) axis ( 14 ). However, the regulation mechanism of circZCCHC14 in the process of accommodation from cartilage into cartilage is still inadequate. N6-methyladenosine (m 6 A) is involved in almost all fundamental aspects of RNA metabolism, which is the most widely distributed RNA modification in eukaryotes ( 15 , 16 ). It has been reported that m 6 A modification exists widely in circRNAs and shows cell specificity. Xu et al . ( 17 ) determined that circZKSCAN1/miR-1208 derived from MSCs regulates m 6 A modification during chondrogenic differentiation. Combined with our previous study, we wondered whether the m 6 A methylation level affects circZCCHC14 on chondrogenic differentiation, as well as which m 6 A methylation modification enzyme plays a critical role. In our present study, we predicted the m 6 A methylation modification enzymes of circZCCHC14 and evaluated their role in regulating the PBMSCs chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Our present study might provide a novel insight into the mechanism function of circZCCHC14 on chondrogenic differentiation. Materials and methods Experimental pigs and sample collection Pigs are very similar to humans in the aspects of anatomical structure, physiology, biochemical, metabolism, and the development mechanism of diseases. The genetic spectrum is also similar to the human gene spectrum. Therefore, using pigs as the experiment model can fully meet the research process of various human pathological development and physiology analyses needs. Earlier studies have shown that Diannan small-ear (DSE) pigs are good cartilage-defect animal models ( 18 ). Therefore, we employed DSE pigs in our present study. Twelve DSE pigs (40 ± 3 days) with similar weaning weights were raised in commercial cages, and bred under the same nutritional and 12:12 light/dark cycle at room temperature. They were offered an ad libitum water. All the animals were provided by Kunming medical university, and the animal experiments were carried out by the Guide to Nursing and Use of Experimental Animals and approved by the Ethics Review Committee of Animal Experiments of Kunming Medical University (kmmu20221858). All animal methods are reported by ARRIVE guidelines. The DSE pigs were divided into Control and Model groups randomly. The pigs in the Model group underwent the Hulth procedure in the right knee joint following the detailed method reported by Jia et al . ( 19 ). To establish a joint injury model in the short term, pigs were released from the cages and driven to run back and forth on the 30-m-long road for 10–15 min every three days. At 6–8 months (15 − 20kg), all the DSE pigs were executed after inhalation anesthesia by isoflurane, and the samples of blood, joint liquid, and tissues at the joint were harvested and stored at − 80°C for further use. Some of the tissues were fixed by 4% polymerization formaldehyde. Isolation of pig PBMSCs The cells in bone tissues were collected and treated with PBS rinse and centrifugation. PBMSCs were isolated from pigs in the Model group according to the previous literature ( 18 , 20 ). PBMSCs were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37 ℃, 5% CO 2 cell culture incubator for 0 days (NC), 7 days (Induced-7d), and 14 days (Induced-14d), IL-1β, TNF-α, and IL-6 stimulation group (Inflammation), Induced for 14 days and then stimulated with IL-1β, TNF-α, and IL-6 (Inflammation + Induced-14d) groups. Vector construction and transfection BMSCs cells induced for 14 days were seeded into 6-well plates with 6×10 5 cells per well and incubated overnight. Cells were transfected with overexpression plasmid of miR-181a, circZCCHC14, FTO, and vector. The small interfering (si) RNA of FTO (si-FTO) and its negative control (NC) were synthesized by GenePharma (Shanghai, China). All the transfections were conducted following the instruction of Lipofectamine 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 80–90% confluent. Two days after transfection, cells were harvested for the following experiments. Real-time quantitative PCR (qPCR) Total RNA in peripheral blood and joint lipids were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression levels of circZCCHC14, FTO, collagen type II alpha 1 chain (COL-2A1), aggrecan (ACAN), GREM1, and miR-181a were measured using qPCR. The primers were designed and synthesized by Sangon (Shanghai, China; Table S1 ). SYBR Green PCR master mix (Applied Biosystems, cat. #4309155) was used to perform qPCR. Then, the productions were conducted on an ABI 7900 system (Foster City, CA, USA) and calculated using the 2 –ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the reference gene. All experiments were tripled. Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) The MeRIP-qPCR was performed as previously described ( 21 ). The Magna methylated RIP (MeRIP) kit (Millipore, cat. #CR203146) was employed. Cells were harvested and then centrifugated at 1,500 rpm at 4℃ for 5 min. After removing the supernatant, cells were mixed with RIP lysis buffer and incubated with the lysate. m 6 A antibody (5 µg) containing magnetic beads was administrated for a rotation of 30 min. The beads were eluted using RIP buffer twice. After rotation overnight, the beads were washed using a high-salt buffer. The RNA enrichment was calculated by qPCR. Western blotting The western blotting analysis was performed on cells and transfections. The proteins in cells were lysis by RIPA lysis buffer (#R0278, Sigma) and quantified by BCA Protein Assay Kit (Beyotime, Jiangsu, China). The standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used. The membranes were blocked with 5% non-fat milk and incubated with the primary antibodies of YTH N6-methyladenosine RNA binding protein (YTHD) F1 (YTHDF1; # ab220162, 1:1000, Abcam), YTHDF2(#ab220163, 1:1000, Abcam), YTHDF3(#ab220161, 1:1000, Abcam), YTHDC1(#ab259990, 1:1000, Abcam), YTHDC2(#ab220160, 1:1000, Abcam), methyltransferase-like 3 (METTL3) (#ab195352, 1:1000, Abcam), METTL14(#ab220030, 1:1000, Abcam), WTAP(#ab195380, 1:1000, Abcam), FTO(#ab126605, 1:20000, Abcam), COL-2A1(#ab188570, 1:1000, Abcam), ACAN(#ab3778, 1:1, Abcam), GREM1(#ab231065, 1:5, Abcam), bone morphogenetic protein 2 (BMP2; # ab214821, 1:1000, Abcam), and GAPDH (#ab9485, 1:2500; Abcam). Then, the PVDF membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody (1:5000). The bands were observed under an ECL kit (Millipore, Germany). Alcian blue staining Firstly, the cells are cleaned with PBS for 10 min twice, fixed with 10% formaldehyde for 20 minutes, and buried with paraffin. Then, the sections were dewaxed, distilled, and soaked with Alley’s new blue stain solution (Sigma, A3157) for 30 min. Finally, the sections were washed three times and observed under the microscope. Bioinformatics Analysis Before the function investigation and verification of the FTO m 6 A-forming and demethylase enzymes on circZCCHC14, bioinformatic analyses were performed. The potential m 6 A modification sites and the m 6 A-forming and demethylase enzymes of circZCCHC14 were predicted in m6Avar and SRAMP databases. RNA pull-down analysis RNA pull-down assay was conducted to determine the interaction between circZCCHC14 and FTO. Briefly, the FTO or FTO-Mut probe was synthesized and biotinylated by GenePharma (Shanghai, China). RNA pull-down assay was carried out using the Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol. The Streptavidin-coupled magnetic beads were incubated with an FTO probe (Servicebio, China). Cells were lysed in a specif ic lysis buffer (Ambion, Austin, TX), treated with magnetic beads, and then washed with precooled lysis buffer and salt buffer solution. In the end, the RNA-binding protein complexes were washed and eluted for real-time qPCR analysis. RNA immunoprecipitation (RIP) assay One EZ-Magna RIP kit (Millipore, Billerica, MA, USA) was used to detect the interaction between circZCCHC14 and FTO according to the manufacturer’s introduction. Generally, the transfected cells were treated with RIP buffer and incubated with anti-Argonaute 2 (Ago2; 1:50; #ab186733) overnight. IgG (1:100; ab109489; Abcam) employed as control. Then, the samples were digested with proteinase K, and TRIzol-chloroform was employed to extract the immunoprecipitated RNA. Finally, the RNAs precipitated by Ago2 or IgG were detected by qPCR. Statistical analysis GraphPad Prism 8 (San Diego, CA, USA) was employed for the statistical analysis. Biorender (Biorender-Javashuo) was used to draw the summary diagram. One-way ANONA comparison analysis was performed on groups. All the experiments were tripled. Data are exhibited as mean ± standard deviation (SD). P < 0.05 was considered a significant difference. Results CircZCCHC14 expression and the m 6 A methylation level were increased in joint injury DSE pigs Firstly, we tested the expression of circZCCHC14 and the m 6 A methylation modification level in joint-damaged DSE pigs. The expression level of circZCCHC14 was significantly higher in the Model group compared with that in the Control groups in both joint liquid and BMSCs of DSE pigs ( P < 0.001; Figs. 1 A, B). The relative enrichment of anti-m 6 A was significantly increased in the Model group compared with the Control group ( P < 0.001; Fig. 1 C), showing a significantly promoted m 6 A methylation level in joint injury DSE pigs. Therefore, we wondered that whether the changes in the level of m 6 A methylation are one of the reasons that affect the expression level of circZCCCHC14. The bioinformatics analysis on circZCCHC14 was performed based on m6Avar and SRAMP databases. Genes of YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, METTL3, METTL14, WTAP, and FTO were identified to be m 6 A methylation modifying enzymes of circZCCHC14. Then, we further analyzed their expression levels in the Model and Control groups by western blotting. Western blotting results showed that the m 6 A methylation modification enzyme YTHDF1 was upregulated and FTO downregulated in the Model group (Fig. 1 D). Therefore, the results summarized that circZCCHC14 and m 6 A methylation levels were highly increased in joint injury DSE pigs, which might be caused by YTHDF1 or FTO m 6 A methylation modification enzymes. m 6 A methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation To investigate the relationship between circZCCHC14 and m 6 A methylation modification levels during the process of cartilage differentiation, in vitro experiments were conducted. The chondrocyte-specific biomarkers of COL-2A1 and ACAN were significantly upregulated at Induced-7d and Induced-14d when compared with Control by both qPCR and western blotting analyses ( P < 0.05, P < 0.01, P < 0.001; Figs. 2 A, B). Moreover, their expression levels raised with the treatment time increased. Alcian blue staining proved the chondrogenic differentiation ability of cells induced by BMSCs at 14 days (Fig. 2 C). Moreover, circZCCHC14 inhibited the differentiation ability (Fig. 2 D). These results showed that the cartilage induction and differentiation effect are obvious, and the cells are successfully induced for further experiments. The expression levels of circZCCHC14, miR-181a, and GREM1, as well as the level of m 6 A methylation were measured by qPCR and MeRIP-qPCR. As shown in Figs. 2 E-G, the expression level of circZCCHC14 and GREM1 were significantly downregulated, miR-181a increased, and m 6 A methylation level decreased in Induced-7d and Induced-14d groups when compared with NC ( P < 0.01, P < 0.001). The western blotting analysis was consistent with that of qPCR in terms of GREM1 , one of the target genes of circZCCHC14 (Fig. 2 H). As demonstrated by our previous study, GREM1 and BMP2 have antagonistic effects, and that they jointly regulate the chondrogenic differentiation of BMSCs. In our present study, BMP2 is upregulated in Induced-7d and Induced-14d compared with NC (Fig. 2 H). The m 6 A-methylation modification enzymes of METTL3 and FTO were highly expressed in cells induced by BMSCs at 7 days and 14 days (Fig. 2 I). The opposite regulation trend between METTL3, FTO and m 6 A methylation might indicated the demethylase role of METTL3 and FTO on m 6 A methylation of circZCCHC14. The above results suggest that the degree of chondalization has increased with the extension of BMSCs cartilage induction and differentiation. Moreover, the level of m 6 A methylation which might be demethylated by FTO or METTL3 was proportional to the expression of circZCCCHC14. FTO was decreased, circZCCHC14 and m 6 A methylation level were increased under inflammatory conditions Inflammation response can exacerbate joint destruction and pain, thus performing an essential role in OA pathogenesis ( 22 ). While, BMSCs are supposed to inhibit inflammation, differentiate into chondrogenic, and relieve pain. Therefore, our study tried to elucidate the role of circZCCHC14 and m 6 A methylation under inflammatory conditions and cartilage differentiation conditions. The qPCR and MeRIP-qPCR tests found that the expression level of circZCCHC14 was significantly increased under inflammation conditions, which was reversed by BMSCs cartilage differentiation ( P < 0.05, P < 0.001, Figs. 3 A, B). As one of the m 6 A methylation modification enzymes, the expression level of METTL3 was increased in the Induced-14d and Inflammation + Induced-14d groups when compared with the Inflammation and NC groups (Fig. 3 C). The expression level of FTO was decreased in the Inflammation group and increased in the Induced group when compared with NC. The BMSCs cartilage differentiation and inflammation treatments showed opposed functions on regulating the expression level of FTO, which provided more evidence of FTO as one demethylated enzyme of circZCCHC14 m 6 A methylation. However, the role between FTO and m 6 A methylation need further confirmation. Then, we measured the expression levels of miR-181a and GREM1 in NC, Inflammation, Induced-14d, and Inflammation + Induced14d. The results showed that miR-181a was significantly decreased under inflammation treatment compared with NC, which can be significantly reversed by Induced-14d ( P < 0.05, P < 0.001, Fig. 3 D), showing an opposite expression trend with that of circZCCHC14 in four groups. As experimented by qPCR and western blotting, the expression level of GREM1 was significantly elevated in Inflammation, and then decreased by Inflammation + Induced-14d ( P < 0.05, P < 0.001, Fig. 3 D, E), being opposed with miR-181a. While, as the other target gene of miR-181a, BMP2 showed a consistent expression trend with that of miR-181a, and opposed with that of GREM1 (Fig. 3 E), showing antagonistic effects with GREM1. The expression levels of chondrocyte-specific biomarkers (COL-2A1 and ACAN) were significantly lower in the Inflammation + Induced-14d group than Induced-14d group ( P < 0.001; Figs. 3 F, G), showing a suppression role of inflammation in cartilage differentiation. Alcian blue staining exhibited that the chondrogenic differentiation degree was much higher in Induced-14d than in NC and Inflammation groups, as well as in Inflammation + Induced-14d (Fig. 3 H). The above experiments have further explained that the expression level of circZCCHC14 was subsequently increased with the m 6 A methylation level induced by inflammation environments, while BMSCs cartilage differentiation can be one essential factor for reversing the elevated level of m 6 A methylation. FTO is one essential m 6 A demethylase enzyme of circZCCHC14 Based on the experiments mentioned above, FTO was considered to be one key m 6 A demethylase enzyme of circZCCHC14, which needed to be further verified. Therefore, RNA pull-down and RIP analyses were conducted to identify the FTO binding role of circZCCHC14. A biotin-coupled probe pull-down assay was then performed and the result showed FTO was detected in the circZCCHC14 pulled-down pellet (Fig. 4 A). RIP assay revealed that circZCCHC14 expression was significantly enriched by anti-FTO compared with the control IgG antibody ( P < 0.001, Fig. 4 B). These results confirmed that FTO is one essential m 6 A demethylase enzyme of circZCCHC14. m 6 A demethylase enzyme FTO regulated the expression levels of circZCCHC14 After confirming the relationship between FTO and m6A methylation, how it regulates circZCCHC14/miR-181a/GREM1 and BMP2 was explored. As our previous study has reported, the axis of circZCCHC14/miR-181a/GREM1 on regulating the chondrogenic differentiation ability ( 14 ), we tried to further identify how FTO m 6 A demethylase influences circZCCHC14/miR-181a/GREM1. We constructed the transfections of si-FTO and then induced by BMSCs cartilage differentiation for 14 days. The expression level of FTO was significantly decreased in a si-FTO + Induced-14d group compared with si-NC + Induced-14d ( P < 0.001; Figs. 5 A, B), showing the success of transfection construction. Compared with si-NC + Induced-14d, the expression levels of chondrocyte-specific biomarkers (COL-2A1 and ACAN) were significantly decreased in the si-FTO + Induced-14d group ( P < 0.001, Figs. 5 C, D). Alcian blue staining experiment showed that the chondrogenic differentiation degree was significantly weakened in si-FTO writer and reader groups compared with si-NC + Induced-14d. Figure 5 F showed that the expression level of circZCCHC14 was significantly elevated after interference with FTO ( P < 0.001). The MeRIP-qPCR result showed a significant increase in m 6 A methylation modification levels in the si-FTO + Induced-14d group compared with the si-NC + Induced-14d group ( P < 0.001, Fig. 5 G). The expression levels of miR-181a and its target genes of GREM1 and BMP2 were tested by qPCR and western blotting experiments. miR-181a was significantly decreased and GREM1 was significantly increased in si-FTO + Induced-14d group compared with si-NC + Induced-14d group ( P < 0.001, Figs. 5 H, I). BMP2 exhibited an opposite expression trend with that of GREM1 in si-FTO + Induced-14d and si-NC + Induced-14d groups (Fig. 5 I). m 6 A demethylase enzyme FTO positively regulated the expression of miR-181a After verifying the relationship between FTO and circZCCHC14, we tried to explore that of FTO and miR-181a by establishing transfections of miR-ctrl + si-NC, miR-181a + si-NC, miR-ctrl + si-FTO, and miR-181a + si-FTO. Firstly, the expression levels of FTO and miR-181a were measured in transfections. Compared with the miR-ctrl + si-NC group, miR-181a was highly expressed when being overexpressed, and FTO was lowly expressed when being interfered with ( P < 0.01, P < 0.001, Figs. 6 A, B), indicating the successful of transfections. Figures 6 C and D showed that the expression levels of COL-2A1 and ACAN were significantly increased in single transfection by overexpressed miR-181a (miR-181a + si-NC, P < 0.001), and decreased in single transfection by interference of FTO (miR-ctrl + si-FTO, P < 0.001). However, when the si-FTO plasmid was transfected with miR-181a vector, the highly expressed COL-2A1 and ACAN were significantly reversed, suggesting that miR-181a can promote the expression of chondrocyte-specific biomarkers by promoting FTO expression. The MeRIP-qPCR experiment results showed that the m 6 A methylation level was significantly reduced and increased by miR-181a + si-NC and miR-ctrl + si-FTO, respectively ( P < 0.01, P < 0.001; Fig. 6 E). Similarly, overexpressed miR-181a and si-FTO showed interactive functions in regulating m 6 A methylation levels. As shown in Figs. 6 F-H, the expression levels of circZCCHC14 and GREM1 exhibited the same regulated trends as that of m 6 A methylation, being significantly promoted by si-FTO and suppressed by overexpressed miR-181a ( P < 0.05, P < 0.01, P < 0.001). As the other target gene of miR-181a, the expression level of BMP2 in four groups was opposed to that of GREM1 (Fig. 6 H). Taken together, the m 6 A demethylase enzyme FTO positively regulated the expression of miR-181a. FTO m 6 A demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis We tested the role of FTO on m 6 A methylation and circZCCHC14/miR-181a/GREM1 by establishing transfections of overexpressed circZCCHC14 and FTO. Firstly, the transfection effect was measured, and the result showed that the expression levels of FTO and circZCCHC14 were both raised when overexpressed, showing a successful transfection ( P < 0.01, P < 0.001, Figs. 7 A, B). Figures 7 C and D showed that the expression levels of COL-2A1 and ACAN were significantly increased in single transfection by overexpressed FTO ( P < 0.001), and decreased in single transfection by overexpressed circZCCHC14 ( P < 0.001). However, when transfected both together, their expression levels were reversed when compared with single transfections, implying that FTO can reverse the increase of COL-2A1 and ACAN induced by circ-ZCCHC14 overexpression. They also showed interactive roles in regulating m 6 A methylation, miR-181a, GREM1, and BMP2 ( P < 0.01, P < 0.001, Figs. 7 E-H). As shown in Figs. 7 E-H, overexpressed FTO decreased m 6 A methylation level, increased miR-181a expression level, raised BMP2 expression level, and reduced that of GREM1. Taken together, the FTO m 6 A demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Discussion CircRNAs are a class of noncoding RNAs that are involved in chondrogenic differentiation, and m 6 A broadly exists in circRNAs. In our present study, we investigated the m 6 A modification enzyme in regulating the BMSCs chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Through this study, we can draw the following results: ( 1 ) CircZCCHC14 expression and the m 6 A methylation level were increased in joint injury DSE pigs; ( 2 ) m 6 A methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation; ( 3 ) FTO was decreased, circZCCHC14 and m6A methylation level were increased under inflammatory conditions; ( 4 ) FTO is one essential m 6 A demethylase enzyme of circZCCHC14; ( 5 ) FTO m 6 A demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis (Fig. 8 ). In our present study, circZCCHC14 was upregulated in joint injury DSE pigs, which is consistent with our previous study ( 14 ). Some other circRNAs were also identified to be involved in chondrogenic differentiation. For example, circATRNL1 promotes chondrogenic differentiation of BMSCs, which is mediated by miR-338-3p ( 23 ). Exosome-transported circRNA_0001236 suppressed cartilage degradation and enhanced chondrogenesis through miR-3677-3p/SRY-box transcription factor 9 (Sox9) axis ( 24 ). In our present study, we found that circZCCHC14 regulates chondrogenic differentiation ability through miR-181a/GREM1 and BMP2 axis. BMP2 belongs to the transforming growth factor β superfamily and has the highest efficiency in inducing chondrogenic differentiation of BMSCs ( 25 ). GREM1 is highly expressed in OA samples, associated with severe knee OA ( 26 , 27 ). In addition, it was also found to be highly in IL-1β-induced chondrocytes, promoting chondrocyte apoptosis and extracellular matrix degradation ( 28 ). As our previous study demonstrated, GREM1 and BMP2 have antagonistic effects, and they jointly regulate the chondrogenic differentiation of PBMSCs ( 14 ), which was consistent with that in BMSCs determined in our present study. Our present study confirmed the role of circZCCHC14/miR-181a/GREM1 and BMP2 in the chondrogenic differentiation of PBMSCs. m 6 A is a transcriptional modification of ncRNAs ( 29 ), the modification of which affects the biological functions of circRNAs ( 30 ). Our present study showed that the m 6 A methylation level was increased in joint injury models. Moreover, the level of m 6 A methylation which might be demethylated by FTO was the reason for the dysregulated expression of circZCCCHC14. Therefore, we investigated how m 6 A methylation influences the expression level of circZCCHC14. Only FTO was identified as the m6A demethylase enzyme of circZCCHC14 after verifying. FTO is the first gene contributing to common forms of human obesity ( 31 ). As reported, FTO has efficient oxidative demethylation activity targeting the abundant m 6 A residues in RNA ( 32 ). Abnormal levels of m 6 A during FTO elevation attenuate cell cycle progression, disrupt functional stem cell differentiation and normal lineage commitment, leading to immune deficiency, neurogenesis retardation, and sterility ( 33 – 35 ). Xu et al . ( 17 ) demonstrated that circZKSCAN1 derived from MSCs regulates m 6 A modification during chondrogenic differentiation by inhibiting miR-1208/FTO. Wang et al . ( 36 ) revealed that RNA m 6 A demethylase FTO promotes OA through demethylating runx2 mRNA and inhibiting OA differentiation. Overexpressed FTO alleviates OA by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis ( 37 ). Yang et al . ( 38 ) demonstrated that FTO-mediated m 6 A demethylation downregulated AC008 transcription, while lower FTO expression led to upregulation of AC008 transcription in OA. Liu et al. revealed that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, providing fresh insights into the therapeutic strategies for OA ( 39 ). In our present study, we confirmed the role of FTO as the m 6 A demethylase enzyme of circZCCHC14 involved in the regulation of chondrogenic differentiation. Considering the essential role of inflammation in OA, we tried to elucidate how FTO-dependent m6A methylation influences inflammation and BMSCs. We found that BMSCs cartilage differentiation reversed FTO-mediated m 6 A modification in inflammatory conditions. He et al . ( 40 ) demonstrated that METTL3-mediated m 6 A modification has a negative influence on the apoptosis and autophagy of chondrocytes in inflammation. METTL3 actively regulates the secretion of inflammatory factors, chondrocyte apoptosis, and extracellular matrix degradation in OA ( 41 ). METTL3-YTHDF2-mediated m 6 A methylation is involved in osteogenic differentiation not only under inflammatory conditions but also under physiological conditions ( 42 ). So far, few studies have demonstrated the FTO-dependent m 6 A methylation in regulating chondrogenic differentiation under inflammatory conditions in OA. However, the association between FTO and inflammation have been studied in other diseases. For examples, Dubey et al. ( 43 ) demonstrated that FTO suppression is associated with myocardial inflammation in vivo analysis. FTO is involved in inflammatory response of LPS-induced septic shock and the regulation of FTO is promising for the treatment of septic shock ( 44 ). Our present study provided novel insight into the FTO m 6 A demethylase on chondrogenic differentiation in inflammatory conditions. Conlusion In summary, our data demonstrate that circZCCHC14 dysregulated by FTO-regulated m 6 A methylation, thus dysregulating the expression of miR-181a/GREM1, providing insights into the mechanism of circZCCHC14 in promoting chondrogenic differentiation of Diannan small-ear pigs PBMSCs. Our present study provided a novel insight into the mechanism function of circZCCHC14 on chondrogenic differentiation. Declarations Funding This work was supported by Yunnan Revitalization Talent Support Program (No.XDYC-QNRC-2022-0318) and Yunnan Medical Reserve Talent Project (No. H-2019013). Competing interests The authors declare no competing interests. Author contributions All authors contributed equally to this work. D.H.Z., H.C. and J.Z. participated in the acquisition of the data. J.Z., B.Z. contributed to data analysis. J.Z.and D.H.Z. revised the manuscript carefully for important content. D.H.Z. and H.C. conceived, designed, and led the study. C.R.W. and J.Z. made contributions to drafting the manuscript. All authors read and approved the final manuscript. Data Availability All data supporting the findings of this study are contained within the article. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Kunming Medical University (The mechanism research of circRNA-ZCCHC14 regulating miR-181a and inhibiting the expression of GREM1 to promoting chondrogenic differentiation of PBMSC. kmmu20221858.02-28-2022) Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish The authors affirm that human research participants provided informed consent for publication of the images in all Figure References Arden N, Nevitt MC. Osteoarthritis: epidemiology. Best practice research Clinical rheumatology. 2006;20(1):3-25. Chimutengwende-Gordon M, Donaldson J, Bentley G. 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Circ_DHRS3 positively regulates GREM1 expression by competitively targeting miR-183-5p to modulate IL-1β-administered chondrocyte proliferation, apoptosis and ECM degradation. International Immunopharmacology. 2021;91:107293. Kumari R, Ranjan P, Suleiman ZG, Goswami SK, Li J, Prasad R, et al. mRNA modifications in cardiovascular biology and disease: with a focus on m6A modification. Cardiovascular research. 2022;118(7):1680-92. Chen R-X, Chen X, Xia L-P, Zhang J-X, Pan Z-Z, Ma X-D, et al. N 6-methyladenosine modification of circNSUN2 facilitates cytoplasmic export and stabilizes HMGA2 to promote colorectal liver metastasis. Nature communications. 2019;10(1):4695. Loos R, Bouchard C. FTO: the first gene contributing to common forms of human obesity. Obesity reviews. 2008;9(3):246-50. Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al. N 6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nature chemical biology. 2011;7(12):885-7. Xiang Y, Laurent B, Hsu C-H, Nachtergaele S, Lu Z, Sheng W, et al. RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature. 2017;543(7646):573-6. Smemo S, Tena JJ, Kim K-H, Gamazon ER, Sakabe NJ, Gómez-Marín C, et al. Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature. 2014;507(7492):371-5. Su R, Dong L, Li C, Nachtergaele S, Wunderlich M, Qing Y, et al. R-2HG exhibits anti-tumor activity by targeting FTO/m6A/MYC/CEBPA signaling. Cell. 2018;172(1):90-105. e23. Wang J, Fu Q, Yang J, Liu J-L, Hou S-M, Huang X, et al. RNA N6-methyladenosine demethylase FTO promotes osteoporosis through demethylating Runx2 mRNA and inhibiting osteogenic differentiation. Aging. 2021;13(17):21134. Cai D, Zhang J, Yang J, Lv Q, Zhong C. Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis. International Immunopharmacology. 2023;114:109524. Yang J, Zhang M, Yang D, Ma Y, Tang Y, Xing M, et al. m6A-mediated Upregulation of AC008 Promotes Osteoarthritis Progression through the miR-328-3p‒AQP1/ANKH axis. Experimental molecular medicine. 2021;53(11):1723-34. Liu W, Jiang T, Zheng W, Zhang J, Li A, Lu C, et al. FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression. Arthritis Research Therapy. 2023;25(1):1-17. He Y, Wang W, Luo P, Wang Y, He Z, Dong W, et al. Mettl3 regulates hypertrophic differentiation of chondrocytes through modulating Dmp1 mRNA via Ythdf1-mediated m6A modification. Bone. 2022;164:116522. Liu Q, Li M, Jiang L, Jiang R, Fu B. METTL3 promotes experimental osteoarthritis development by regulating inflammatory response and apoptosis in chondrocyte. Biochemical biophysical research communications. 2019;516(1):22-7. Zhang Y, Gu X, Li D, Cai L, Xu Q. METTL3 regulates osteoblast differentiation and inflammatory response via Smad signaling and MAPK signaling. International Journal of Molecular Sciences. 2019;21(1):199. Dubey PK, Patil M, Singh S, Dubey S, Ahuja P, Verma SK, et al. Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice. Molecular Cellular Biochemistry. 2022:1-13. Luo J, Wang F, Sun F, Yue T, Zhou Q, Yang C, et al. Targeted inhibition of FTO demethylase protects mice against LPS-induced septic shock by suppressing NLRP3 inflammasome. Frontiers in immunology. 2021;12:663295. Supplementary Files AuthorChecklistFull.pdf WBimages.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4334272","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":302998666,"identity":"3c04a87c-4f34-48eb-8a33-662e2dea4a0b","order_by":0,"name":"Daohong Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACefbGBuMfPDZ2/OwNRGox7DncUMwgk5Ys2XOAWGtupDd8ZrA5zLjhRgKROhh7DjZuLshhZja4+XjjDYYam2iCWtjZG5uNZ5xh45O8nVZswXAsLbeBCFvaDHh7eJj5bueYSTA2HCasheFGYvsP3n9AxTfPEK+lwZiHx4Bxwg0eIrUY9hxsMJzBkwAMZKBfEojxizx7+wODDzz/gVF5eOONDzU2RDgMCRhIJJCiHKKFVB2jYBSMglEwMgAAx0lDV4IwRj0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2969-8575","institution":"The second affiliated hospital of Kunming medicial university","correspondingAuthor":true,"prefix":"","firstName":"Daohong","middleName":"","lastName":"Zhao","suffix":""},{"id":302998667,"identity":"89ce2eb0-5700-4d70-bcd5-c509c5d9120a","order_by":1,"name":"Bo Zhao","email":"","orcid":"","institution":"the second people's hospital of baoshan city","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zhao","suffix":""},{"id":302998668,"identity":"c00df84d-ad6f-44ea-bfb8-b4f4aa549c9b","order_by":2,"name":"Jia Zhong","email":"","orcid":"","institution":"the people's hospital of xishuangbanna state","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Zhong","suffix":""},{"id":302998669,"identity":"1b2ebc18-c91e-4378-974e-7af308328e24","order_by":3,"name":"Hong Chen","email":"","orcid":"","institution":"the first people's hospital of kunming city","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Chen","suffix":""},{"id":302998670,"identity":"0627f0fe-c01c-4a4e-bd04-c1a357c2f2f8","order_by":4,"name":"Jun Zhang","email":"","orcid":"","institution":"the second affiliated hospital of kunming medicial university","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""},{"id":302998671,"identity":"ee188122-7489-49c9-9603-dfc32f76b676","order_by":5,"name":"Chaoran Wang","email":"","orcid":"","institution":"the second affiliated hospital of kunming medicial university","correspondingAuthor":false,"prefix":"","firstName":"Chaoran","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-04-27 13:34:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4334272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4334272/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57090935,"identity":"85cd74b9-52ba-4900-9348-e54ab9ea99f4","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircZCCHC14 expression and the m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA methylation level were increased in joint injury DSE pigs. \u003c/strong\u003e(A, B) QPCR analysis tested the expression level of circZCCHC14 in Control and Model groups in both joint liquid and BMSCs samples. (C) MeRIP-qPCR analysis was performed on measuring the level of m\u003csup\u003e6\u003c/sup\u003eA methylation in Control and Model groups. (D) Western blotting analysis was performed on measuring the expression levels of m\u003csup\u003e6\u003c/sup\u003eA-methylation modification enzymes in Control and Model groups. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/1a2f6cad4dabd3c74b822ade.png"},{"id":57091407,"identity":"0b4c5aa9-1116-4cbe-87db-9816ee73a3a7","added_by":"auto","created_at":"2024-05-24 13:07:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation.\u003c/strong\u003e (A) qPCR analysis on testing the expression level of chondrocyte-specific biomarkers of \u003cem\u003eCOL-2A1\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e in NC, Induced-7d, and Induced-14d groups. (B) Western blotting analysis on testing the expression level of chondrocyte-specific biomarkers of \u003cem\u003eCOL-2A1\u003c/em\u003e and \u003cem\u003eACAN\u003c/em\u003e in NC, Induced-7d, and Induced-14d groups. (C, D) Alcian blue staining on showing the chondrogenic differentiation ability of cells induced by BMSCs. (E) qPCR analysis on verifying the expression of circZCCHC14. (F) MeRIP-qPCR analysis on verifying the expression of m\u003csup\u003e6\u003c/sup\u003eA methylation level. (G) qPCR analysis on verifying the expression of miR-181a and GREM1. (H) Western blotting analysis on verifying the expression of GREM1, BMP2, METTL3, FTO, and GAPDH. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/c7a88f5d562d9eef6f20d4be.png"},{"id":57090938,"identity":"99f99c47-16f4-4dda-a408-fa471578bd64","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTO was decreased, circZCCHC14 and m6A methylation level were increased under inflammatory conditions. \u003c/strong\u003e(A) qPCR measured the expression level of circZCCHC14 in NC, Inflammation, Induced-14d, and Inflammation +Induced-14d groups. (B) MeRIP-qPCR measured the level of m\u003csup\u003e6\u003c/sup\u003eA methylation in NC, Inflammation, Induced-14d, and Inflammation +Induced-14d groups. (C) The expression levels of m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzymes (METTL3 and FTO) were tested by western blotting. (D) qPCR experiment analyzed the expression levels of miR-181a and GREM1. (E) Western blotting analysis was conducted to evaluate the expression levels of GREM1 and BMP2. (F) The expression levels of chondrocyte-specific biomarkers of COL-2A1 and ACAN were tested by qPCR. (G) The expression levels of chondrocyte-specific biomarkers of COL-2A1 and ACAN were tested by western blotting. (H) Alcian blue staining shows the chondrogenic differentiation ability in NC, Inflammation, Induced-14d, and Inflammation +Induced-14d groups. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/8cc86f9541017c7935bf297f.png"},{"id":57091408,"identity":"1076a9d4-9138-4761-b285-18e49cada18a","added_by":"auto","created_at":"2024-05-24 13:07:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTO is one essential m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA demethylase enzyme of circZCCHC14.\u003c/strong\u003e (A) The RNA pull-down assay on identifying the target enzyme of FTO on circZCCHC14. (B) The RIP assay was conducted to verify the interaction role between FTO and circZCCHC14. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/e7c924f4c867085913f5955a.png"},{"id":57090941,"identity":"a18e9050-17f6-4177-a6f3-60673c6de76e","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA demethylase enzyme FTO regulated the expression levels of circZCCHC14. \u003c/strong\u003e(A, B) qPCR and western blotting analyses on testing the expression level of FTO after being treated with si-FTO under BMSCs cartilage differentiation for 14 days. (C, D) qPCR and western blotting analyses on testing the expression level of chondrocyte-specific biomarkers (COL-2A1 and ACAN) after being treated with si-FTO under BMSCs cartilage differentiation for 14 days. (E) Alcian blue staining shows the chondrogenic differentiation ability in si-NC+Induced-14d, si-writer+Induced-14, and si-reader+Induced-14d. (F) qPCR analysis on verifying the expression of circZCCHC14. (G) MeRIP-qPCR analysis on verifying the m\u003csup\u003e6\u003c/sup\u003eA methylation level. (H) qPCR analysis on testing the expression of miR-181a and GREM1. (I) Western blotting analysis on evaluating the expression of GREM1 and BMP2. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/67d8327a11143d9325db58e0.png"},{"id":57090940,"identity":"f9b08282-66e0-4431-8a00-f045cbb25397","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA demethylase enzyme FTO positively regulated the expression of miR-181a. \u003c/strong\u003e(A) QPCR analysis on testing the expression level of FTO and miR-181a after being treated with si-FTO and overexpressed miR-181a under BMSCs cartilage differentiation for 14 days. (B) Western blotting analysis on evaluating the expression level of FTO. (C, D) represented the qPCR and western blotting analyses on testing the expression level of chondrocyte-specific biomarkers (COL-2A1 and ACAN) after being treated with si-FTO and overexpressed miR-181a under BMSCs cartilage differentiation for 14 days. (E) MeRIP-qPCR analysis was performed to assess the m\u003csup\u003e6\u003c/sup\u003eA methylation level. (F, G) qPCR analysis was conducted on verifying the expression of circZCCHC14, GREM1, and BMP2. (H) Western blotting analysis on evaluating the expression of GREM1 and BMP2. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/7168788d196f1496a8cdec1d.png"},{"id":57090944,"identity":"684fe549-eb51-4220-8e14-b0336428604e","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTO m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis.\u003c/strong\u003e (A) qPCR analysis was conducted on testing the expression level of FTO and circZCCHC14 after being treated with overexpressed FTO and circZCCHC14. (B) Western blotting analysis was performed on evaluating the expression level of FTO. (C, D) qPCR and western blotting analyses on testing the expression level of chondrocyte-specific biomarkers (COL-2A1 and ACAN) after being treated with overexpressed FTO and circZCCHC14. (E) MeRIP-qPCR analysis on measuring the m\u003csup\u003e6\u003c/sup\u003eA methylation level. (F) qPCR analysis on verifying the expression of miR-181a. (G, H) represented the qPCR and western blotting analyses on verifying the expression of GREM1 and BMP2. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/7bcf131e07287894c90c254e.png"},{"id":57090942,"identity":"9357ab63-afe9-45cc-9ccd-6d4ccbe35c7a","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":32380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration for the working model of circZCCHC14. \u003c/strong\u003eFTO m\u003csup\u003e6\u003c/sup\u003eA demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in bone marrow mesenchymal stem cells (BMSCs) chondrogenic differentiation of Diannan small ear pigs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/9f8bff50543ecb1203005c04.png"},{"id":57747402,"identity":"8f3f2e74-7ae5-40e4-b792-2e6d4a4f6939","added_by":"auto","created_at":"2024-06-05 06:06:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1528973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/6c262cbb-5fb4-41b1-a685-3e1cd8842420.pdf"},{"id":57091409,"identity":"3c3c8f5d-bd8d-4673-8321-8111f49a1ef8","added_by":"auto","created_at":"2024-05-24 13:07:35","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":107453,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/0009e323409a280f2ffe3f0f.pdf"},{"id":57090945,"identity":"042dd8c9-d5df-48e3-8c93-dfb81098f162","added_by":"auto","created_at":"2024-05-24 12:59:35","extension":"pptx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2404146,"visible":true,"origin":"","legend":"","description":"","filename":"WBimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4334272/v1/2a06a87b108541964806892b.pptx"}],"financialInterests":"","formattedTitle":"FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is one of the joint function defect diseases, with no effective clinical therapies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The treatment of articular cartilage defects has always been a major challenge that joint surgeons urgently need to solve. Clinical treatment methods consist of drug-conservative treatment, joint cleaning, grinding and shaping surgery, microfracture surgery, and autologous cartilage transplantation, all of which showed limited therapeutic effects and cannot repair articular cartilage defects completely (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent years, the development of tissue engineering technology has opened up new avenues for the treatment of cartilage defects (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Seed cells, scaffold materials, and growth-inducing factors were three key factors of tissue engineering technology and have been a hot study topic in recent years.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs) have become ideal seed cells for cartilage tissue engineering due to their multidirectional differentiation ability, easy cultivation, and expansion characteristics (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), among which bone marrow mesenchymal stem cells (BMSCs) were commonly used (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). How to induce and regulate the directional differentiation of MSCs into chondrocytes is currently an important research issue in cartilage tissue engineering. Currently, the relevant regulatory mechanisms of MSCs chondrogenic differentiation are not fully understood. Therefore, exploring the relevant regulatory mechanisms of MSCs chondrogenic differentiation may provide support for exploring more effective differentiation-promoting strategies.\u003c/p\u003e \u003cp\u003eCircular RNA (circRNA), mainly located in the cytoplasm and exosomes, is not affected by RNA external enzymes, thus showing more stable and conserved features than other ncRNAs, which have been identified as potential noninvasive biomarkers and as drugs for the OA treatment (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In recent years, some studies have confirmed that circRNAs have important functions in the maintenance and differentiation of stem cells (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). It has also been recognized in studying the growth and differentiation of cells, the directional development of tissue organs, and the determination of disease treatment targets (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). As reported, circRNAs have played an important regulatory role in bone and cartilage differentiation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). For example, circCREBBP actives the Smad1/5 pathway through the transforming growth factor beta 2 (TGFβ2)/activin receptor-like kinases 1(ALK1) axis, thus modulating cartilage degradation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Our previous study revealed the role of circZCCHC14 in affecting the chondrogenic differentiation ability of peripheral blood‑derived mesenchymal stem cells (PBMSCs) through the miR-181a/Gremlin 1(GREM1) axis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, the regulation mechanism of circZCCHC14 in the process of accommodation from cartilage into cartilage is still inadequate.\u003c/p\u003e \u003cp\u003eN6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) is involved in almost all fundamental aspects of RNA metabolism, which is the most widely distributed RNA modification in eukaryotes (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). It has been reported that m\u003csup\u003e6\u003c/sup\u003eA modification exists widely in circRNAs and shows cell specificity. Xu \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) determined that circZKSCAN1/miR-1208 derived from MSCs regulates m\u003csup\u003e6\u003c/sup\u003eA modification during chondrogenic differentiation. Combined with our previous study, we wondered whether the m\u003csup\u003e6\u003c/sup\u003eA methylation level affects circZCCHC14 on chondrogenic differentiation, as well as which m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzyme plays a critical role.\u003c/p\u003e \u003cp\u003eIn our present study, we predicted the m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzymes of circZCCHC14 and evaluated their role in regulating the PBMSCs chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Our present study might provide a novel insight into the mechanism function of circZCCHC14 on chondrogenic differentiation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental pigs and sample collection\u003c/h2\u003e \u003cp\u003ePigs are very similar to humans in the aspects of anatomical structure, physiology, biochemical, metabolism, and the development mechanism of diseases. The genetic spectrum is also similar to the human gene spectrum. Therefore, using pigs as the experiment model can fully meet the research process of various human pathological development and physiology analyses needs. Earlier studies have shown that Diannan small-ear (DSE) pigs are good cartilage-defect animal models (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, we employed DSE pigs in our present study. Twelve DSE pigs (40\u0026thinsp;\u0026plusmn;\u0026thinsp;3 days) with similar weaning weights were raised in commercial cages, and bred under the same nutritional and 12:12 light/dark cycle at room temperature. They were offered an \u003cem\u003ead libitum\u003c/em\u003e water. All the animals were provided by Kunming medical university, and the animal experiments were carried out by the Guide to Nursing and Use of Experimental Animals and approved by the Ethics Review Committee of Animal Experiments of Kunming Medical University (kmmu20221858). All animal methods are reported by ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eThe DSE pigs were divided into Control and Model groups randomly. The pigs in the Model group underwent the Hulth procedure in the right knee joint following the detailed method reported by Jia \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). To establish a joint injury model in the short term, pigs were released from the cages and driven to run back and forth on the 30-m-long road for 10\u0026ndash;15 min every three days.\u003c/p\u003e \u003cp\u003eAt 6\u0026ndash;8 months (15\u0026thinsp;\u0026minus;\u0026thinsp;20kg), all the DSE pigs were executed after inhalation anesthesia by isoflurane, and the samples of blood, joint liquid, and tissues at the joint were harvested and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further use. Some of the tissues were fixed by 4% polymerization formaldehyde.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of pig PBMSCs\u003c/h2\u003e \u003cp\u003eThe cells in bone tissues were collected and treated with PBS rinse and centrifugation. PBMSCs were isolated from pigs in the Model group according to the previous literature (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). PBMSCs were cultured with Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e cell culture incubator for 0 days (NC), 7 days (Induced-7d), and 14 days (Induced-14d), IL-1β, TNF-α, and IL-6 stimulation group (Inflammation), Induced for 14 days and then stimulated with IL-1β, TNF-α, and IL-6 (Inflammation\u0026thinsp;+\u0026thinsp;Induced-14d) groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVector construction and transfection\u003c/h2\u003e \u003cp\u003eBMSCs cells induced for 14 days were seeded into 6-well plates with 6\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. Cells were transfected with overexpression plasmid of miR-181a, circZCCHC14, FTO, and vector. The small interfering (si) RNA of FTO (si-FTO) and its negative control (NC) were synthesized by GenePharma (Shanghai, China). All the transfections were conducted following the instruction of Lipofectamine 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 80\u0026ndash;90% confluent. Two days after transfection, cells were harvested for the following experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReal-time quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA in peripheral blood and joint lipids were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression levels of circZCCHC14, FTO, collagen type II alpha 1 chain (COL-2A1), aggrecan (ACAN), GREM1, and miR-181a were measured using qPCR. The primers were designed and synthesized by Sangon (Shanghai, China; \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). SYBR Green PCR master mix (Applied Biosystems, cat. #4309155) was used to perform qPCR. Then, the productions were conducted on an ABI 7900 system (Foster City, CA, USA) and calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the reference gene. All experiments were tripled.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMethylated RNA immunoprecipitation qPCR (MeRIP-qPCR)\u003c/h2\u003e \u003cp\u003eThe MeRIP-qPCR was performed as previously described (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The Magna methylated RIP (MeRIP) kit (Millipore, cat. #CR203146) was employed. Cells were harvested and then centrifugated at 1,500 rpm at 4℃ for 5 min. After removing the supernatant, cells were mixed with RIP lysis buffer and incubated with the lysate. m\u003csup\u003e6\u003c/sup\u003eA antibody (5 \u0026micro;g) containing magnetic beads was administrated for a rotation of 30 min. The beads were eluted using RIP buffer twice. After rotation overnight, the beads were washed using a high-salt buffer. The RNA enrichment was calculated by qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe western blotting analysis was performed on cells and transfections. The proteins in cells were lysis by RIPA lysis buffer (#R0278, Sigma) and quantified by BCA Protein Assay Kit (Beyotime, Jiangsu, China). The standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used. The membranes were blocked with 5% non-fat milk and incubated with the primary antibodies of YTH N6-methyladenosine RNA binding protein (YTHD) F1 (YTHDF1; # ab220162, 1:1000, Abcam), YTHDF2(#ab220163, 1:1000, Abcam), YTHDF3(#ab220161, 1:1000, Abcam), YTHDC1(#ab259990, 1:1000, Abcam), YTHDC2(#ab220160, 1:1000, Abcam), methyltransferase-like 3 (METTL3) (#ab195352, 1:1000, Abcam), METTL14(#ab220030, 1:1000, Abcam), WTAP(#ab195380, 1:1000, Abcam), FTO(#ab126605, 1:20000, Abcam), COL-2A1(#ab188570, 1:1000, Abcam), ACAN(#ab3778, 1:1, Abcam), GREM1(#ab231065, 1:5, Abcam), bone morphogenetic protein 2 (BMP2; # ab214821, 1:1000, Abcam), and GAPDH (#ab9485, 1:2500; Abcam). Then, the PVDF membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody (1:5000). The bands were observed under an ECL kit (Millipore, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAlcian blue staining\u003c/h2\u003e \u003cp\u003eFirstly, the cells are cleaned with PBS for 10 min twice, fixed with 10% formaldehyde for 20 minutes, and buried with paraffin. Then, the sections were dewaxed, distilled, and soaked with Alley\u0026rsquo;s new blue stain solution (Sigma, A3157) for 30 min. Finally, the sections were washed three times and observed under the microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics Analysis\u003c/h2\u003e \u003cp\u003eBefore the function investigation and verification of the FTO m\u003csup\u003e6\u003c/sup\u003eA-forming and demethylase enzymes on circZCCHC14, bioinformatic analyses were performed. The potential m\u003csup\u003e6\u003c/sup\u003eA modification sites and the m\u003csup\u003e6\u003c/sup\u003eA-forming and demethylase enzymes of circZCCHC14 were predicted in m6Avar and SRAMP databases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA pull-down analysis\u003c/h2\u003e \u003cp\u003eRNA pull-down assay was conducted to determine the interaction between circZCCHC14 and FTO. Briefly, the FTO or FTO-Mut probe was synthesized and biotinylated by GenePharma (Shanghai, China). RNA pull-down assay was carried out using the Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol. The Streptavidin-coupled magnetic beads were incubated with an FTO probe (Servicebio, China). Cells were lysed in a specif ic lysis buffer (Ambion, Austin, TX), treated with magnetic beads, and then washed with precooled lysis buffer and salt buffer solution. In the end, the RNA-binding protein complexes were washed and eluted for real-time qPCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP) assay\u003c/h2\u003e \u003cp\u003eOne EZ-Magna RIP kit (Millipore, Billerica, MA, USA) was used to detect the interaction between circZCCHC14 and FTO according to the manufacturer\u0026rsquo;s introduction. Generally, the transfected cells were treated with RIP buffer and incubated with anti-Argonaute 2 (Ago2; 1:50; #ab186733) overnight. IgG (1:100; ab109489; Abcam) employed as control. Then, the samples were digested with proteinase K, and TRIzol-chloroform was employed to extract the immunoprecipitated RNA. Finally, the RNAs precipitated by Ago2 or IgG were detected by qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 (San Diego, CA, USA) was employed for the statistical analysis. Biorender (Biorender-Javashuo) was used to draw the summary diagram. One-way ANONA comparison analysis was performed on groups. All the experiments were tripled. Data are exhibited as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCircZCCHC14 expression and the m\u003csup\u003e6\u003c/sup\u003eA methylation level were increased in joint injury DSE pigs\u003c/h2\u003e \u003cp\u003eFirstly, we tested the expression of circZCCHC14 and the m\u003csup\u003e6\u003c/sup\u003eA methylation modification level in joint-damaged DSE pigs. The expression level of circZCCHC14 was significantly higher in the Model group compared with that in the Control groups in both joint liquid and BMSCs of DSE pigs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The relative enrichment of anti-m\u003csup\u003e6\u003c/sup\u003eA was significantly increased in the Model group compared with the Control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), showing a significantly promoted m\u003csup\u003e6\u003c/sup\u003eA methylation level in joint injury DSE pigs. Therefore, we wondered that whether the changes in the level of m\u003csup\u003e6\u003c/sup\u003eA methylation are one of the reasons that affect the expression level of circZCCCHC14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe bioinformatics analysis on circZCCHC14 was performed based on m6Avar and SRAMP databases. Genes of YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, METTL3, METTL14, WTAP, and FTO were identified to be m\u003csup\u003e6\u003c/sup\u003eA methylation modifying enzymes of circZCCHC14. Then, we further analyzed their expression levels in the Model and Control groups by western blotting. Western blotting results showed that the m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzyme YTHDF1 was upregulated and FTO downregulated in the Model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Therefore, the results summarized that circZCCHC14 and m\u003csup\u003e6\u003c/sup\u003eA methylation levels were highly increased in joint injury DSE pigs, which might be caused by YTHDF1 or FTO m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003em\u003csup\u003e6\u003c/sup\u003eA methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between circZCCHC14 and m\u003csup\u003e6\u003c/sup\u003eA methylation modification levels during the process of cartilage differentiation, \u003cem\u003ein vitro\u003c/em\u003e experiments were conducted. The chondrocyte-specific biomarkers of COL-2A1 and ACAN were significantly upregulated at Induced-7d and Induced-14d when compared with Control by both qPCR and western blotting analyses (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Moreover, their expression levels raised with the treatment time increased. Alcian blue staining proved the chondrogenic differentiation ability of cells induced by BMSCs at 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Moreover, circZCCHC14 inhibited the differentiation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results showed that the cartilage induction and differentiation effect are obvious, and the cells are successfully induced for further experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of circZCCHC14, miR-181a, and GREM1, as well as the level of m\u003csup\u003e6\u003c/sup\u003eA methylation were measured by qPCR and MeRIP-qPCR. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G, the expression level of circZCCHC14 and GREM1 were significantly downregulated, miR-181a increased, and m\u003csup\u003e6\u003c/sup\u003eA methylation level decreased in Induced-7d and Induced-14d groups when compared with NC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The western blotting analysis was consistent with that of qPCR in terms of \u003cem\u003eGREM1\u003c/em\u003e, one of the target genes of circZCCHC14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). As demonstrated by our previous study, GREM1 and BMP2 have antagonistic effects, and that they jointly regulate the chondrogenic differentiation of BMSCs. In our present study, BMP2 is upregulated in Induced-7d and Induced-14d compared with NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The m\u003csup\u003e6\u003c/sup\u003eA-methylation modification enzymes of METTL3 and FTO were highly expressed in cells induced by BMSCs at 7 days and 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). The opposite regulation trend between METTL3, FTO and m\u003csup\u003e6\u003c/sup\u003eA methylation might indicated the demethylase role of METTL3 and FTO on m\u003csup\u003e6\u003c/sup\u003eA methylation of circZCCHC14. The above results suggest that the degree of chondalization has increased with the extension of BMSCs cartilage induction and differentiation. Moreover, the level of m\u003csup\u003e6\u003c/sup\u003eA methylation which might be demethylated by FTO or METTL3 was proportional to the expression of circZCCCHC14.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFTO was decreased, circZCCHC14 and m\u003csup\u003e6\u003c/sup\u003eA methylation level were increased under inflammatory conditions\u003c/h2\u003e \u003cp\u003eInflammation response can exacerbate joint destruction and pain, thus performing an essential role in OA pathogenesis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). While, BMSCs are supposed to inhibit inflammation, differentiate into chondrogenic, and relieve pain. Therefore, our study tried to elucidate the role of circZCCHC14 and m\u003csup\u003e6\u003c/sup\u003eA methylation under inflammatory conditions and cartilage differentiation conditions. The qPCR and MeRIP-qPCR tests found that the expression level of circZCCHC14 was significantly increased under inflammation conditions, which was reversed by BMSCs cartilage differentiation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs one of the m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzymes, the expression level of METTL3 was increased in the Induced-14d and Inflammation\u0026thinsp;+\u0026thinsp;Induced-14d groups when compared with the Inflammation and NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The expression level of FTO was decreased in the Inflammation group and increased in the Induced group when compared with NC. The BMSCs cartilage differentiation and inflammation treatments showed opposed functions on regulating the expression level of FTO, which provided more evidence of FTO as one demethylated enzyme of circZCCHC14 m\u003csup\u003e6\u003c/sup\u003eA methylation. However, the role between FTO and m\u003csup\u003e6\u003c/sup\u003eA methylation need further confirmation. Then, we measured the expression levels of miR-181a and \u003cem\u003eGREM1\u003c/em\u003e in NC, Inflammation, Induced-14d, and Inflammation\u0026thinsp;+\u0026thinsp;Induced14d. The results showed that miR-181a was significantly decreased under inflammation treatment compared with NC, which can be significantly reversed by Induced-14d (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), showing an opposite expression trend with that of circZCCHC14 in four groups. As experimented by qPCR and western blotting, the expression level of GREM1 was significantly elevated in Inflammation, and then decreased by Inflammation\u0026thinsp;+\u0026thinsp;Induced-14d (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), being opposed with miR-181a. While, as the other target gene of miR-181a, BMP2 showed a consistent expression trend with that of miR-181a, and opposed with that of GREM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), showing antagonistic effects with GREM1. The expression levels of chondrocyte-specific biomarkers (COL-2A1 and ACAN) were significantly lower in the Inflammation\u0026thinsp;+\u0026thinsp;Induced-14d group than Induced-14d group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G), showing a suppression role of inflammation in cartilage differentiation. Alcian blue staining exhibited that the chondrogenic differentiation degree was much higher in Induced-14d than in NC and Inflammation groups, as well as in Inflammation\u0026thinsp;+\u0026thinsp;Induced-14d (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The above experiments have further explained that the expression level of circZCCHC14 was subsequently increased with the m\u003csup\u003e6\u003c/sup\u003eA methylation level induced by inflammation environments, while BMSCs cartilage differentiation can be one essential factor for reversing the elevated level of m\u003csup\u003e6\u003c/sup\u003eA methylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFTO is one essential m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14\u003c/h2\u003e \u003cp\u003eBased on the experiments mentioned above, FTO was considered to be one key m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14, which needed to be further verified. Therefore, RNA pull-down and RIP analyses were conducted to identify the FTO binding role of circZCCHC14. A biotin-coupled probe pull-down assay was then performed and the result showed FTO was detected in the circZCCHC14 pulled-down pellet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). RIP assay revealed that circZCCHC14 expression was significantly enriched by anti-FTO compared with the control IgG antibody (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results confirmed that FTO is one essential m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003em\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme FTO regulated the expression levels of circZCCHC14\u003c/h2\u003e \u003cp\u003eAfter confirming the relationship between FTO and m6A methylation, how it regulates circZCCHC14/miR-181a/GREM1 and BMP2 was explored. As our previous study has reported, the axis of circZCCHC14/miR-181a/GREM1 on regulating the chondrogenic differentiation ability (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), we tried to further identify how FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase influences circZCCHC14/miR-181a/GREM1. We constructed the transfections of si-FTO and then induced by BMSCs cartilage differentiation for 14 days. The expression level of FTO was significantly decreased in a si-FTO\u0026thinsp;+\u0026thinsp;Induced-14d group compared with si-NC\u0026thinsp;+\u0026thinsp;Induced-14d (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), showing the success of transfection construction. Compared with si-NC\u0026thinsp;+\u0026thinsp;Induced-14d, the expression levels of chondrocyte-specific biomarkers (COL-2A1 and ACAN) were significantly decreased in the si-FTO\u0026thinsp;+\u0026thinsp;Induced-14d group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Alcian blue staining experiment showed that the chondrogenic differentiation degree was significantly weakened in si-FTO writer and reader groups compared with si-NC\u0026thinsp;+\u0026thinsp;Induced-14d. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF showed that the expression level of circZCCHC14 was significantly elevated after interference with FTO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The MeRIP-qPCR result showed a significant increase in m\u003csup\u003e6\u003c/sup\u003eA methylation modification levels in the si-FTO\u0026thinsp;+\u0026thinsp;Induced-14d group compared with the si-NC\u0026thinsp;+\u0026thinsp;Induced-14d group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The expression levels of miR-181a and its target genes of GREM1 and BMP2 were tested by qPCR and western blotting experiments. miR-181a was significantly decreased and GREM1 was significantly increased in si-FTO\u0026thinsp;+\u0026thinsp;Induced-14d group compared with si-NC\u0026thinsp;+\u0026thinsp;Induced-14d group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, I). BMP2 exhibited an opposite expression trend with that of GREM1 in si-FTO\u0026thinsp;+\u0026thinsp;Induced-14d and si-NC\u0026thinsp;+\u0026thinsp;Induced-14d groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003em\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme FTO positively regulated the expression of miR-181a\u003c/h2\u003e \u003cp\u003eAfter verifying the relationship between FTO and circZCCHC14, we tried to explore that of FTO and miR-181a by establishing transfections of miR-ctrl\u0026thinsp;+\u0026thinsp;si-NC, miR-181a\u0026thinsp;+\u0026thinsp;si-NC, miR-ctrl\u0026thinsp;+\u0026thinsp;si-FTO, and miR-181a\u0026thinsp;+\u0026thinsp;si-FTO. Firstly, the expression levels of FTO and miR-181a were measured in transfections. Compared with the miR-ctrl\u0026thinsp;+\u0026thinsp;si-NC group, miR-181a was highly expressed when being overexpressed, and FTO was lowly expressed when being interfered with (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), indicating the successful of transfections. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e showed that the expression levels of COL-2A1 and ACAN were significantly increased in single transfection by overexpressed miR-181a (miR-181a\u0026thinsp;+\u0026thinsp;si-NC, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and decreased in single transfection by interference of FTO (miR-ctrl\u0026thinsp;+\u0026thinsp;si-FTO, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, when the si-FTO plasmid was transfected with miR-181a vector, the highly expressed COL-2A1 and ACAN were significantly reversed, suggesting that miR-181a can promote the expression of chondrocyte-specific biomarkers by promoting FTO expression. The MeRIP-qPCR experiment results showed that the m\u003csup\u003e6\u003c/sup\u003eA methylation level was significantly reduced and increased by miR-181a\u0026thinsp;+\u0026thinsp;si-NC and miR-ctrl\u0026thinsp;+\u0026thinsp;si-FTO, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Similarly, overexpressed miR-181a and si-FTO showed interactive functions in regulating m\u003csup\u003e6\u003c/sup\u003eA methylation levels. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H, the expression levels of circZCCHC14 and GREM1 exhibited the same regulated trends as that of m\u003csup\u003e6\u003c/sup\u003eA methylation, being significantly promoted by si-FTO and suppressed by overexpressed miR-181a (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As the other target gene of miR-181a, the expression level of BMP2 in four groups was opposed to that of GREM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Taken together, the m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme FTO positively regulated the expression of miR-181a.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFTO m\u003csup\u003e6\u003c/sup\u003eA demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis\u003c/h2\u003e \u003cp\u003eWe tested the role of FTO on m\u003csup\u003e6\u003c/sup\u003eA methylation and circZCCHC14/miR-181a/GREM1 by establishing transfections of overexpressed circZCCHC14 and FTO. Firstly, the transfection effect was measured, and the result showed that the expression levels of FTO and circZCCHC14 were both raised when overexpressed, showing a successful transfection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e showed that the expression levels of COL-2A1 and ACAN were significantly increased in single transfection by overexpressed FTO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and decreased in single transfection by overexpressed circZCCHC14 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, when transfected both together, their expression levels were reversed when compared with single transfections, implying that FTO can reverse the increase of COL-2A1 and ACAN induced by circ-ZCCHC14 overexpression. They also showed interactive roles in regulating m\u003csup\u003e6\u003c/sup\u003eA methylation, miR-181a, GREM1, and BMP2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H). As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H, overexpressed FTO decreased m\u003csup\u003e6\u003c/sup\u003eA methylation level, increased miR-181a expression level, raised BMP2 expression level, and reduced that of GREM1. Taken together, the FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCircRNAs are a class of noncoding RNAs that are involved in chondrogenic differentiation, and m\u003csup\u003e6\u003c/sup\u003eA broadly exists in circRNAs. In our present study, we investigated the m\u003csup\u003e6\u003c/sup\u003eA modification enzyme in regulating the BMSCs chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Through this study, we can draw the following results: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) CircZCCHC14 expression and the m\u003csup\u003e6\u003c/sup\u003eA methylation level were increased in joint injury DSE pigs; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) m\u003csup\u003e6\u003c/sup\u003eA methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) FTO was decreased, circZCCHC14 and m6A methylation level were increased under inflammatory conditions; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) FTO is one essential m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn our present study, circZCCHC14 was upregulated in joint injury DSE pigs, which is consistent with our previous study (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Some other circRNAs were also identified to be involved in chondrogenic differentiation. For example, circATRNL1 promotes chondrogenic differentiation of BMSCs, which is mediated by miR-338-3p (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Exosome-transported circRNA_0001236 suppressed cartilage degradation and enhanced chondrogenesis through miR-3677-3p/SRY-box transcription factor 9 (Sox9) axis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In our present study, we found that circZCCHC14 regulates chondrogenic differentiation ability through miR-181a/GREM1 and BMP2 axis. BMP2 belongs to the transforming growth factor β superfamily and has the highest efficiency in inducing chondrogenic differentiation of BMSCs (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). GREM1 is highly expressed in OA samples, associated with severe knee OA (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In addition, it was also found to be highly in IL-1β-induced chondrocytes, promoting chondrocyte apoptosis and extracellular matrix degradation (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). As our previous study demonstrated, GREM1 and BMP2 have antagonistic effects, and they jointly regulate the chondrogenic differentiation of PBMSCs (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), which was consistent with that in BMSCs determined in our present study. Our present study confirmed the role of circZCCHC14/miR-181a/GREM1 and BMP2 in the chondrogenic differentiation of PBMSCs.\u003c/p\u003e \u003cp\u003em\u003csup\u003e6\u003c/sup\u003eA is a transcriptional modification of ncRNAs (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), the modification of which affects the biological functions of circRNAs (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Our present study showed that the m\u003csup\u003e6\u003c/sup\u003eA methylation level was increased in joint injury models. Moreover, the level of m\u003csup\u003e6\u003c/sup\u003eA methylation which might be demethylated by FTO was the reason for the dysregulated expression of circZCCCHC14. Therefore, we investigated how m\u003csup\u003e6\u003c/sup\u003eA methylation influences the expression level of circZCCHC14. Only FTO was identified as the m6A demethylase enzyme of circZCCHC14 after verifying. FTO is the first gene contributing to common forms of human obesity (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). As reported, FTO has efficient oxidative demethylation activity targeting the abundant m\u003csup\u003e6\u003c/sup\u003eA residues in RNA (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Abnormal levels of m\u003csup\u003e6\u003c/sup\u003eA during FTO elevation attenuate cell cycle progression, disrupt functional stem cell differentiation and normal lineage commitment, leading to immune deficiency, neurogenesis retardation, and sterility (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Xu \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) demonstrated that circZKSCAN1 derived from MSCs regulates m\u003csup\u003e6\u003c/sup\u003eA modification during chondrogenic differentiation by inhibiting miR-1208/FTO. Wang \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) revealed that RNA m\u003csup\u003e6\u003c/sup\u003eA demethylase FTO promotes OA through demethylating runx2 mRNA and inhibiting OA differentiation. Overexpressed FTO alleviates OA by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Yang \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) demonstrated that FTO-mediated m\u003csup\u003e6\u003c/sup\u003eA demethylation downregulated AC008 transcription, while lower FTO expression led to upregulation of AC008 transcription in OA. Liu et al. revealed that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, providing fresh insights into the therapeutic strategies for OA (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In our present study, we confirmed the role of FTO as the m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14 involved in the regulation of chondrogenic differentiation.\u003c/p\u003e \u003cp\u003eConsidering the essential role of inflammation in OA, we tried to elucidate how FTO-dependent m6A methylation influences inflammation and BMSCs. We found that BMSCs cartilage differentiation reversed FTO-mediated m\u003csup\u003e6\u003c/sup\u003eA modification in inflammatory conditions. He \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) demonstrated that METTL3-mediated m\u003csup\u003e6\u003c/sup\u003eA modification has a negative influence on the apoptosis and autophagy of chondrocytes in inflammation. METTL3 actively regulates the secretion of inflammatory factors, chondrocyte apoptosis, and extracellular matrix degradation in OA (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). METTL3-YTHDF2-mediated m\u003csup\u003e6\u003c/sup\u003eA methylation is involved in osteogenic differentiation not only under inflammatory conditions but also under physiological conditions (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). So far, few studies have demonstrated the FTO-dependent m\u003csup\u003e6\u003c/sup\u003eA methylation in regulating chondrogenic differentiation under inflammatory conditions in OA. However, the association between FTO and inflammation have been studied in other diseases. For examples, Dubey et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) demonstrated that FTO suppression is associated with myocardial inflammation \u003cem\u003ein vivo\u003c/em\u003e analysis. FTO is involved in inflammatory response of LPS-induced septic shock and the regulation of FTO is promising for the treatment of septic shock (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Our present study provided novel insight into the FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase on chondrogenic differentiation in inflammatory conditions.\u003c/p\u003e"},{"header":"Conlusion","content":"\u003cp\u003eIn summary, our data demonstrate that circZCCHC14 dysregulated by FTO-regulated m\u003csup\u003e6\u003c/sup\u003eA methylation, thus dysregulating the expression of miR-181a/GREM1, providing insights into the mechanism of circZCCHC14 in promoting chondrogenic differentiation of Diannan small-ear pigs PBMSCs. Our present study provided a novel insight into the mechanism function of circZCCHC14 on chondrogenic differentiation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Yunnan Revitalization Talent Support Program (No.XDYC-QNRC-2022-0318) and Yunnan Medical Reserve Talent Project (No. H-2019013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed equally to this work. D.H.Z., H.C. and J.Z. participated in the acquisition of the data. J.Z., B.Z. contributed to data analysis. J.Z.and D.H.Z. revised the manuscript carefully for important content. D.H.Z. and H.C. conceived, designed, and led the study. C.R.W. and J.Z. made contributions to drafting the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Kunming Medical University (The mechanism research of circRNA-ZCCHC14 regulating miR-181a and inhibiting the expression of GREM1 to promoting chondrogenic differentiation of PBMSC. kmmu20221858.02-28-2022)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that human research participants provided informed consent for publication of the images in all Figure\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArden N, Nevitt MC. Osteoarthritis: epidemiology. Best practice research Clinical rheumatology. 2006;20(1):3-25.\u003c/li\u003e\n\u003cli\u003eChimutengwende-Gordon M, Donaldson J, Bentley G. 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Targeted inhibition of FTO demethylase protects mice against LPS-induced septic shock by suppressing NLRP3 inflammasome. Frontiers in immunology. 2021;12:663295.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"circZCCHC14, m6A methylation, chondrogenic differentiation, Bone marrow mesenchymal stem cells, FTO","lastPublishedDoi":"10.21203/rs.3.rs-4334272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4334272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCircular RNAs (circRNAs) are a class of noncoding RNAs that are involved in chondrogenic differentiation, and N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) broadly exists in circRNAs.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eA joint injury model was constructed on Diannan small-ear (DSE) pigs. Transfections were constructed using Lipofectamine 2000. Real-time quantitative PCR (qPCR), Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR), and western blotting analyses were performed. Alcian blue staining tested the chondrogenic differentiation ability. The potential m\u003csup\u003e6\u003c/sup\u003eA methylation modification enzymes and sites of circZCCHC14 were predicted in m6Avar and SRAMP databases. RNA pull-down and RIP assays were conducted to determine the interaction between circZCCHC14 and FTO.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCircZCCHC14 expression and the m\u003csup\u003e6\u003c/sup\u003eA methylation level were increased in joint injury DSE pigs. m\u003csup\u003e6\u003c/sup\u003eA methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation. FTO was decreased, circZCCHC14 and m\u003csup\u003e6\u003c/sup\u003eA methylation level were increased under inflammatory conditions. FTO is one essential m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme of circZCCHC14. m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme FTO regulated the expression levels of circZCCHC14. m\u003csup\u003e6\u003c/sup\u003eA demethylase enzyme FTO positively regulated the expression of miR-181a. FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data showed the physiological significance of FTO m\u003csup\u003e6\u003c/sup\u003eA demethylase in regulating axis circZCCHC14/miR-181a/GREM1 and BMP2, providing a potentially effective therapeutic target for the treatment of joint injury or Osteoarthritis (OA).\u003c/p\u003e","manuscriptTitle":"FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 12:59:30","doi":"10.21203/rs.3.rs-4334272/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c724742-a775-45e0-bc87-891d8cb05115","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-05T06:06:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-24 12:59:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4334272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4334272","identity":"rs-4334272","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}