Abstract
Background
The role of N6-methyladenosine (m6A)-modified circRNAs in disease progression is of great significance. However, the specific impact of m6A modification of circSTAT6 on osteoporosis (OP) is still uncertain.
Methods
The qRT-PCR was employed to assess the levels of METTL3, circSTAT6, miR-188-3p, and Beclin1. To investigate the interaction between miR-188-3p and circSTAT6 or Beclin, a dual-luciferase reporter assay was performed. To evaluate osteogenic differentiation in bone marrow mesenchymal stem cell (BMSC), western blot analysis was conducted to evaluate the protein expression of osteogenic markers, including ALP, OPN, and Runx2. In addition, alizarin red and alkaline phosphatase (ALP) staining assays were employed to assess osteogenesis.
Results
The findings revealed that the downregulation of circSTAT6 was observed in OP. On the other hand, the overexpression of circSTAT6 was found to enhance the osteogenic differentiation of BMSC. In addition, the involvement of METTL3 in mediating m6A methylation of circSTAT6 was identified, which ultimately promoted osteogenesis. Furthermore, circSTAT6 functioned as an miR-188-3p sponge to regulate the expression of Beclin1. Further study revealed that the osteogenic-enhancing effect caused by circSTAT6 overexpression was counteracted by introducing a miR-188-3p mimic. Similarly, the osteogenic-promoting impact of the miR-188-3p inhibitor was reversed by suppressing Beclin1 expression.
Conclusions
The present study revealed, for the first time, that METTL3-mediated m6A modification of circSTAT6 regulated the miR-188-3p/Beclin1 axis to promote the osteogenic differentiation of BMSC. These findings offer a potential therapeutic target for the treatment of OP.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13018-025-05720-4.
Keywords: METTL3, m6A modification, circSTAT6, miR-188-3p, Osteoporosis
Introduction
Osteoporosis (OP) is a common bone disease characterized by low bone mass and increased susceptibility to fractures [1]. While it can affect individuals of any age, it is more commonly observed in postmenopausal women and older men [2]. The consequences of OP can be severe, often leading to disability and even death among elderly patients [3, 4]. Although there are treatment options available, such as calcitonin, estrogen and bisphosphonates, these can only offer palliative care as there is currently no cure for OP [5]. Given the challenges presented, it is imperative to promptly identify effective and beneficial strategies for OP treatment.
Bone-marrow mesenchymal stem cells (BMSC) have the ability to self-renew and differentiate into different cell types such as osteoblasts, adipocytes and chondrocytes [6, 7]. They play an important role in tissue regeneration, thus making them extensively used in cell therapy approaches [8, 9]. One of the contributing factors to the development of OP is the reduced capacity of BMSC to differentiate into osteoblasts [10–12]. However, the precise mechanism underlying the osteogenic differentiation of BMSCs still requires further investigation.
CircRNAs, which are non-coding RNAs, have a crucial role in regulating the onset and progression of various diseases [13, 14]. They are involved in controlling a diverse array of biological activities [15, 16]. Several studies have also emphasized the significance of circRNA dysregulation in the context of OP. In a specific study, the regulatory role of circRNA_0048211 in mitigating the progression of postmenopausal osteoporosis (PMOP) by influencing the expression of BMP2 through miRNA-93-5p was investigated [17]. Moreover, it has been observed that the inhibition of miRNA-339-5p by circRNA_0001795 can slow down the progression of OP by regulating YAP1 expression [18]. Our analysis of the GSE161361 dataset revealed significant downregulation of circSTAT6 in OP patients compared to healthy controls, prompting investigation into its functional role.
The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes, comprising over 80% of all methylated RNA bases [19]. The m6A modification process includes the installation of m6A ‘writers’, the removal of m6A ‘erasers’, and the recognition of m6A ‘readers‘ [20]. m6A modification has significant implications in various diseases and cellular functions, including OP [21]. METTL3, a crucial m6A ‘writers’, has been implicated in bone metabolism [22]. For instance, METTL3 deficiency in osteoblasts disrupts bone formation, while its overexpression enhances osteogenic differentiation of BMSCs by stabilizing RUNX2 mRNA [23]. Despite these advances, the effect of METTL3-mediated m6A modification on circSTAT6 in OP remains unexplored.
Beclin1, a multifunctional protein, has recently been implicated in bone remodeling and OP progression [24]. Studies demonstrate that Beclin1 deficiency exacerbates bone loss, whereas its overexpression promotes osteogenesis and improves bone mass in preclinical OP models [25]. Bioinformatic analysis further identified miR-188-3p as a potential mediator linking circSTAT6 to Beclin1, suggesting a novel regulatory axis in BMSC differentiation.
Therefore, we proposed that METTL3-mediated m6A modification enhanced circSTAT6 stability, enabling it to sequester miR-188-3p and relieve its repression of Beclin1, thereby promoting BMSC osteogenesis. These findings suggested that circSTAT6, modified by m6A, could be a promising therapeutic target for the treatment of OP.
Materials and methods
Clinical samples
Between 2021 and 2022, we collected a total of 25 specimens of trabecular bone tissue from individuals diagnosed with OP. In addition, a set of 25 patients who were suffering from traumatic fractures were included as negative controls. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University.
Cell culture and transfection
Human bone marrow mesenchymal stem cells (BMSCs) were obtained from the China Center for Type Culture Collection in Wuhan. The BMSC were cultured in modified eagle’s medium (MEM, Gibco, USA). The BMSC were then incubated in a humidified incubator at 37 °C with 5% CO2. The process of osteogenic differentiation was initiated by introducing the BMSCs into a 12-well plate and incubating them with the osteogenic differentiation medium (Cyagen Biosciences, China). The circSTAT6 shRNA, Beclin1 shRNA, miR-188-3p inhibitor, and miR-188-3p mimic were obtained from Ribo (Guangzhou, China). The pcDNA3.1 cricSTAT6 plasmid and pcDNA3.1 METTL3 plasmid were synthesized by Genepharma (Shanghai, China). For cell transfection, Lipofectamine 3000 was employed following the instructions provided by the manufacturer.
qRT-PCR
The extraction of total RNA was performed using TRIzol (Invitrogen) and cDNA synthesis was carried out using the PrimeScriptTM RT reagent Kit. Subsequently, qRT-PCR was conducted using the TB Green Premix Ex TaqTM reageant. Normalization of miRNAs was performed relative to U6, whereas normalization of circRNA and mRNA expression was achieved by comparing it to GAPDH. The primers were listed in supplementary Table e1.
Luciferase reporter assay
The 3’-UTR sequences of circSTAT6 and Beclin1, which contain the putative binding sites for miR-188-3p, were initially amplified using PCR. These amplified fragments were subsequently cloned into the pGL6-miR vector (Genepharma, Shanghai, China). Prior to transfection, 293T cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well and allowed to proliferate for 24 h until they reached 70–80% confluency. Following this, the cells were co-transfected with 100 ng of the pGL6-miR reporter vector and 50 nM of the miR-188-3p mimic (or a negative control mimic) using Lipofectamine 3000. After a 48-hour incubation period, the cells were lysed, and luciferase activities were quantified using the dual-luciferase reporter assay system.
Fluorescence in situ hybridization (FISH) assay
Probes for miR-188-3p and circSTAT6 were obtained and synthesized from GenePharma (Shanghai, China). Following the guidelines provided by the manufacturer, BMSC underwent hybridization with the aforementioned probes at 37 °C overnight. Immunofluorescence microscopy was utilized to capture the resulting images.
RNA Immunoprecipitation (RIP)
To evaluate the enrichment of circSTAT6 and miR-188-3p, we employed the Magna RIP kit. The lysate was subjected to incubation in RIP buffer containing magnetic beads bound to either anti-Ago2 or anti-IgG antibody. After RNA purification, qRT-PCR analysis was performed to assess the expression levels of miR-188-3p and circSTAT6.
Western blot
To analyze the cellular proteins, we utilized the RIPA buffer for the extraction process. Subsequently, the proteins from different samples underwent electrophoresis on an SDS-PAGE gel. The gel was then transferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was exposed to a 1:1000 dilution of primary antibodies, specifically anti-ALP, anti-RUNX2, and anti-OPN (Proteintech), at 4 °C. To visualize the membranes, they were treated with secondary antibodies and captured using Image Lab software.
Methylated RNA Immunoprecipitation (Me-RIP) assay
To investigate the m6A accumulation in circSTAT6, we employed TRIzol reagent for total RNA extraction. Protein A/G magnetic beads were utilized with either anti-m6A antibody or IgG to specifically bind the total RNA. The isolation of m6A-modified RNA was achieved through subsequent washing with elution buffer. Finally, qRT-PCR analysis was performed to detect the m6A levels in circSTAT6.
Cell proliferation
After transfection, the potential for cellular proliferation was assessed by conducting an EdU assay (Beyoutime, China). To carry out the assay, BMSC were exposed to an EdU solution for a period of 30 min, followed by staining the nuclei with a DAPI solution for 5 min. Ultimately, fluorescence microscopy was employed to capture images of the cells.
Alizarin red staining
To examine the calcium deposition in BMSCs, Alizarin Red staining was carried out. Following a 2-week incubation in osteogenic medium, BMSCs were fixed using a 4% formaldehyde solution and subsequently subjected to staining using the alizarin red solution (Beyotime, China) for a duration of 30 min. Quantification was achieved by incubating the BMSCs with a 10% acetic acid for 30 min, followed by the measurement of absorbance at 405 nm using a spectrophotometer.
Alkaline phosphatase (ALP) staining
In short, the ALP staining was conducted with the BCIP/NBT Kit (Beyotime, China) following the guidelines provided by the manufacturer. For the quantification of alkaline phosphatase activity, the ALP Assay Kit (Beyotime, China) was employed. The spectrophotometer was utilized to measure the absorbance at 415 nm. Subsequently, the value was determined by normalizing it to the protein content.
Statistical analysis
Statistical results were showed as mean ± standard deviation (SD). To assess variations among multiple groups, we conducted an analysis of variance (ANOVA). Additionally, a t-test was used to compare differences between two groups. p < 0.05 was defined as statistically significant.
Results
Overexpressing circSTAT6 promoted the osteogenic differentiation of BMSC
The GSE161361 cohort was utilized to screen for differentially expressed circRNAs between OP and healthy samples. The expression of circSTAT6 (has_circ_0027120) was found to be lower in OP samples compared to healthy samples (Fig. 1A). To confirm the downregulation of circSTAT6 in patients with OP, we evaluated its expression in clinical samples. The demographic results indicated that no significant differences were observed in age, height, weight and bone mineral density (BMD) between the two groups. However, the T-score was significantly lower in the osteoporosis group compared to the control group (Table 1). The results of qRT-PCR showed a significant decrease in the expression of circSTAT6 in patients with OP compared to the healthy samples (Fig. 1B). Sanger sequencing was also performed to confirm the back-spliced junction of circSTAT6 (Fig. 1C). Additionally, RNase-R treatment was conducted to validate the presence of circSTAT6, demonstrating its resistance to RNase-R cleavage. In contrast, the levels of linear STAT6 were significantly reduced (Fig. 1D).
Fig. 1.
Overexpressing circSTAT6 promoted the osteogenic differentiation of BMSC. (A) Heatmap of circSTAT6 expression from the GSE161361 dataset. (B) The expression of circSTAT6 in clinical samples. (C) Sanger sequencing confirmed the circular format of circSTAT6. (D) RNase R treatment confirmed the existence of circSTAT6 in BMSC. (E) The circSTAT6 expression was assessed using qRT-PCR after transfection. (F) EdU assay of BMSC after overexpressing circSTAT6. (G, H) The mRNA and protein expressions of RUNX2, ALP and OPN. (I) The images and quantitation of alizarin red staining. (I) The images and quantitation of ALP staining. *P < 0.05, **P < 0.01
Table 1.
Demographic characteristics of clinical samples
| Control (n = 25) | Osteoporosis (n = 25) | P value | |
|---|---|---|---|
| Age | 59.26 ± 2.16 | 60.18 ± 4.32 | 0.072 |
| Weight (Kg) | 57.38 ± 3.72 | 59.20 ± 4.72 | 0.106 |
| Height (cm) | 157 ± 3.21 | 155 ± 2.89 | 0.076 |
| BMD (g/cm2) | 0.90 ± 0.06 | 0.87 ± 0.09 | 0.125 |
| T-score Lumbar Spine (L1-4) | -0.26 ± 0.03 | -2.65 ± 0.12 | <0.0001 |
Next, we transfected the overexpressed circSTAT6 plasmid into BMSC to investigate its effect on the osteogenic differentiation (Fig. 1E). EdU experiments revealed that the overexpression of circSTAT6 had a positive impact on the proliferation of BMSC (Fig. 1F). The qRT-PCR results further confirmed that circSTAT6 overexpression led to an increase in the mRNA levels of osteogenic differentiation markers, including ALP, OPN, and RUNX2 (Fig. 1G). Consistent with the qRT-PCR findings, the protein levels of these markers were found to be upregulated in BMSCs when circSTAT6 was overexpressed (Fig. 1H, Supplementary Fig. 1). Furthermore, the overexpression of circSTAT6 resulted in increased calcium deposition and ALP activity in BMSC (Fig. 1I, J). Overall, our results indicated that circSTAT6 overexpression could enhance the osteogenic differentiation of BMSC.
METTL3 enhanced circSTAT6 expression through m6A modification and promoted the osteogenic differentiation of BMSC
By employing qRT-PCR, we examined the expression of m6A writing enzymes (METTL3, METTL14, and WTAP) in clinical samples. The results revealed a noteworthy decrease in the expression levels of METTL3 and METTL14 mRNA, with METTL3 showing a particularly pronounced reduction, in the OP group (Fig. 2A, B). However, no significant difference in WTAP expression was observed between the OP and control groups (Fig. 2C). As a result, we chose to focus on further investigating METTL3. In order to investigate the potential mechanism underlying the regulation of cirstat6 by METTL3, we performed a transfection experiment using an overexpressed METTL3 plasmid to increase the expression of METTL3 mRNA and protein (Fig. 2D, E). The results obtained from qRT-PCR demonstrated an increase in the expression of circSTAT6 following the overexpression of METTL3 (Fig. 2F). Furthermore, the MeRIP-PCR results demonstrated a noteworthy elevation in the m6A level of circSTAT6 following transfection with OE-METTL3 (Fig. 2G). Moreover, a positive correlation was observed between the expression of METTL3 and circSTAT6 (Fig. 2H). These results revealed that METTL3 enhanced circSTAT6 expression via m6A modification.
Fig. 2.
METTL3 enhanced circSTAT6 expression through m6A modification and promoted the osteogenic differentiation of BMSC. (A-C) The mRNA expressions of m6A writers (METTL3, METTL14 and WTAP) in clinical samples. (D, E) The mRNA and protein expression of METTL3 after transfected overexpressing plasmid. (F) The expression of circSTAT6 in BMSC after overexpressing METTL3. (G) The m6A level of circSTAT6 after overexpression of METTL3 in BMSC. (H) Pearson correlation between METTL3 and circSTAT6 expression. (I, J) The mRNA and protein expressions of RUNX2, ALP and OPN. (K) The images and quantitation of alizarin red staining. (L) The images and quantitation of ALP staining. *P < 0.05, **P < 0.01
The subsequent analysis aimed to investigate the influence of METTL3 on the osteogenic differentiation of BMSC. Through EdU experiments, it was determined that the upregulation of METTL3 had a positive effect on the proliferation of BMSC (Supplementary Fig. 2). In addition, the findings demonstrated that the overexpression of METTL3 led to an increase in both the gene and protein levels of osteogenic differentiation markers (Fig. 2I, J). Furthermore, the overexpression of METTL3 resulted in heightened calcium deposition and ALP activity in BMSC (Fig. 2K, L). Overall, these findings indicated that the augmentation of METTL3 could enhance the osteogenic differentiation of BMSC.
Silencing circSTAT6 abolished the effects of METTL3 on osteogenic differentiation of BMSC
To investigate the impact of circSTAT6 and METTL3 on osteogenic differentiation of BMSC, we conducted transfection experiments on BMSC with METTL3 overexpression and sh-circSTAT6. The qRT-PCR results demonstrated that the upregulation of circSTAT6 caused by METTL3 overexpression was effectively reversed by sh-circSTAT6 (Fig. 3A), indicating the successful transfection process. The results of EdU analysis revealed that the increase in cell proliferation induced by METTL3 overexpression was abolished by circSTAT6 inhibition (Fig. 3B). Additionally, the results obtained from qRT-PCR and western blot experiments indicated that circSTAT6 inhibition also abolished the increases in mRNA and protein levels of osteogenic markers (Fig. 3C, D). As anticipated, the repression of circSTAT6 prevented the increase in calcium deposition and ALP activity induced by overexpressed METTL3 in BMSCs (Fig. 3E, F). Altogether, silencing circSTAT6 abolished the effects of METTL3 on osteogenic differentiation of BMSC.
Fig. 3.
Silencing circSTAT6 abolished the effects of METTL3 on osteogenesis. (A) The circSTAT6 expression was assessed using qRT-PCR after co-transfection. (B) EdU assay of BMSC after co-transfection. (C, D) The mRNA and protein expressions of RUNX2, ALP and OPN. (E) The images and quantitation of alizarin red staining. (F) The images and quantitation of ALP staining. *P < 0.05, **P < 0.01
CircSTAT6 acted as a sponge for miR-188-3p to regulate Beclin1expression
In order to explore the regulatory mechanism of circSTAT6, a comprehensive analysis utilizing bioinformatics was conducted to forecast potential miRNAs binding to circSTAT6. The examination revealed four specific miRNAs that were observed to bind to circSTAT6: miR-1197, miR-1294, miR-188-3p, and miR-658 (Fig. 4A). Subsequently, following the knockout of circSTAT6, the expression levels of the aforementioned four miRNAs were assessed in BMSC. The findings demonstrated that the downregulation of circSTAT6 led to a significant increase in the expression of miR-188-3p and miR-1294, with a particular emphasis on miR-188-3p (Fig. 4B). Thus, it was selected for further study. The binding relationship between circSTAT6 and miR-188-3p was subsequently verified through luciferase reporter and RIP assays (Fig. 4C, D). Furthermore, FISH experiments demonstrated the co-localization of circSTAT6 and miR-188-3p in the cytoplasm of BMSC (Fig. 4E). Target genes of miR-188-3p were predicted using Targetscan, miRDB, and Starbase databases (Fig. 4F). Among the four potential genes, the miR-188-3p mimic significantly reduced the expression of Beclin1 (Fig. 4G). Subsequently, a luciferase reporter gene was employed to identify the interactions between these sites (Fig. 4H). The results of qRT-PCR demonstrated a significant downregulation of Beclin1 mRNA in the OP group compared to the control group (Fig. 4I). Additionally, rescue experiments demonstrated that the inhibitory effect of sh-circSTAT6 on Beclin1 could be counteracted by a miR-188-3p inhibitor (Fig. 4J, K). Collectively, these findings indicated that circSTAT6 acted as a sponge for miR-188-3p to regulate Beclin1expression.
Fig. 4.
CircSTAT6 acted as a sponge for miR-188-3p to regulate Beclin1expression. (A) Bioinformatics prediction of circSTAT6-binding miRNAs using circBank and circInteractome. (B) The expression of four predicted miRNAs after knockdown of circSTAT6 in BMSC. (C, D) The binding relationship was confirmed by luciferase reporter and RIP assays. (E) The locations of miR-188-3p and circSTAT6 were detected using FISH assay. (F) The predicted targeted mRNAs of miR-188-3p. (G) The expressions of four predicted mRNAs were detected using qRT-PCR after inhibiting miR-188-3p in BMSC. (H) The binding relationship was confirmed by luciferase reporter. (I) The expression of Beclin1 in OP sanmples. (J, K) The mRNA and protein expressions of Beclin1 after co-transfection. *P < 0.05, **P < 0.01
MiR-188-3p mimic reversed the promoting effect of circSTAT6 overexpression on osteogenic differentiation of BMSC
To confirm that miR-188-3p was involved in circSTAT6-mediated osteogenesis, we transfected miR-188-3p mimics into circSTAT6-overexpressing BMSC. Overexpression of circSTAT6 could significantly reduce the expression of miR-188-3p, while co-transfection of miR-188-3p mimics could restore the expression of miR-188-3p (Fig. 5A). Consequently, circSTAT6-mediated promotion of cell proliferation was counteracted by miR-188-3p mimics (Fig. 5B). In addition, the overexpression of osteogenic markers induced by circSTAT6 was inhibited when miR-188-3p mimic was introduced (Fig. 5C, D). Similarly, overexpression of miR-188-3p significantly reduced the promotion of calcium deposition (Fig. 5E, Supplementary Fig. 3A) and ALP activity mediated by circSTAT6 (Fig. 5F, Supplementary Fig. 3B). These findings indicated that circSTAT6 regulated the expression of miR-188-3p, thereby promoting osteogenic differentiation in BMSC.
Fig. 5.
MiR-188-3p mimic reversed the promoting effect of circSTAT6 overexpression on osteogenesis. (A) The miR-188-3p expression was assessed using qRT-PCR after co-transfection. (B) EdU assay of BMSC after co-transfection. (C, D) The mRNA and protein expressions of RUNX2, ALP and OPN. (E) The images of alizarin red staining. (F) The images of ALP staining. *P < 0.05, **P < 0.01
Silencing Beclin1 reversed the promoting effect of miR-188-3p inhibitor on BMSC osteogenic differentiation
Before exploring the role of Beclin1, we first established the functional role of the miR-188-3p inhibitor in the osteogenesis of BMSC. The results demonstrated that the miR-188-3p inhibitor significantly enhanced the expression of osteogenic markers at both the mRNA and protein levels (Supplementary Fig. 4). Subsequently, we conducted a functional examination by co-transfecting sh-Beclin1 with the miR-188-3p inhibitor. The assessment of the miR-188-3p inhibitor resulted in an elevation of Beclin1 levels, whereas knockdown of Beclin1 led to a partial decrease in its abundance (Fig. 6A). Consequently, the expression of sh-Beclin1 successfully inhibited the ability of the miR-188-3p inhibitor to enhance cell proliferation (Fig. 6B). In addition, the miR-188-3p inhibitor substantially enhanced the expression of osteogenic markers on both the mRNA and protein levels. However, when co-transfected with sh-Beclin1, the impact of the miR-188-3p inhibitor was partially compromised (Fig. 6C, D). Furthermore, knockdown of Beclin1 also weakened the elevation of calcium deposition induced by the miR-188-3p inhibitor (Fig. 6E, Supplementary Fig. 5A), along with the ALP activity (Fig. 6F, Supplementary Fig. 5B). Altogether, these findings revealed that Silencing Beclin1 reversed the promoting effect of miR-188-3p inhibitor on BMSC osteogenic differentiation.
Fig. 6.
Silencing Beclin1 reversed the promoting effect of miR-188-3p inhibitor on osteogenesis. (A) The Beclin1 expression was assessed using qRT-PCR after co-transfection. (B) EdU assay of BMSC after co-transfection. (C, D) The mRNA and protein expressions of RUNX2, ALP and OPN. (E) The images of alizarin red staining. (F) The images of ALP staining. *P < 0.05, **P < 0.01
Discussion
OP is a chronic skeletal condition characterized by bone loss, abnormalities in microarchitecture, and increased bone fragility [26, 27]. The dysregulation of circRNAs have been extensively studied as a potential diagnostic indicator and treatment target for various human ailments [28, 29]. In particular, several circRNAs have been discovered to play a crucial role in the progression of OP [30, 31]. For example, recent studies have demonstrated that the downregulation of circRNA_0001275 can serve as a diagnostic marker for OP, thus aiding in the clinical diagnosis of OP [32]. However, the role of circSTAT6 on OP progression has not been fully understood. In our study, we discovered that METTL3-mediated m6A modification of circSTAT6 enhanced the osteogenic differentiation of BMSC through the miR-188-3p/Beclin pathway.
This research aimed to investigate the role of circSTAT6 in the osteogenesis process. Initially, we confirmed the reduced expression of circSTAT6 in patients with OP. Furthermore, we discovered that circSTAT6 and its increased levels contribute to the enhancement of osteogenesis. In conjunction with the observed decrease in samples. In conjunction with the decrease in circSTAT6 expression observed in OP samples, our data suggested a potential association between the downregulation of circSTAT6 and impaired osteogenesis in OP.
The alteration of m6A is a key focus in studying the control of gene expression in various physiological and pathological processes [33]. The m6A modification is generated through the collaboration of a methyltransferase complex known as the ‘writer’, which comprises METTL3, METTL14, and WTAP [34]. Previous studies have shown that METTL3 plays a crucial role in regulating the differentiation of BMSC that contribute to bone tissue formation [35]. In present study, we observed that the expression levels of METTL3, which showed the most notable variations, were decreased in individuals with OP. In addition, the study observed a positive influence of METTL3 on bone formation in BMSCs, suggesting that METTL3 may have a role in mitigating the progression of OP.
The role of METTL3-mediated m6A RNA modification in the progression of OP has been demonstrated [36]. One example is the promotion of osteogenic differentiation of BMSCs through the miR-144-3p/BMPR1B axis by METTL3-mediated long-coding RNA LINC00657 [21]. To date, there have been no reports on the limited study period of circRNA catalyzed by METTL3 for m6A modification in mediating OP progression. This study is the first to investigate the role of m6A-modified circRNAs in OP. Our findings demonstrated that METTL3 promoted the osteogenic differentiation of BMSC through m6A methylation of circSTAT6.
The ceRNA theory, which is widely accepted, serves as the underlying mechanism of action for circRNAs [37]. This theory also applies to circSTAT6 in our research. Our investigation demonstrated that circSTAT6 functioned as a sponge for miR-188-3p. In a previous study, it was reported that miR-188-3p has an inhibitory impact on the osteogenic differentiation of human dental pulp stem cells [38]. However, limited research has been conducted on the underlying mechanism of miR-188-3p on osteogenic differentiation of BMSC. miRNAs possess the ability to regulate mRNA expression by forming complementary base pairs at the post-transcriptional level [25, 39–42]. Previous studies have linked Beclin1, a gene targeted by miR-188-3p, to the advancement of OP [25]. In our study, we observed that silencing Beclin1 reversed the promotive effect of the miR-188-3p inhibitor on the osteogenic differentiation of BMSCs. Therefore, we concluded that miR-188-3p played a regulatory role in osteogenesis by targeting Beclin1.
Conclusions
In summary, circSTAT6, facilitated by METTL3, enhanced the osteogenic differentiation of BMSC through the miR-144-3p/Beclin1 axis. These findings suggested that circSTAT6, modified by m6A, could be a promising therapeutic target for the treatment of OP.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
None.
Author contributions
LY and SYB wrote the original draft, WYQ prepared the conceptualization, and CH prepared the review and editing.
Funding
This work was supported by Hubei Provincial Natural Science Foundation (No. 2023AFB936) and The Foundation of Health Commission of Hubei Province (WJ2023F075).
Data availability
The data are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yue Luo and Yubo Shi contributed equally to this work.
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Data Availability Statement
The data are available from the corresponding author upon reasonable request.