METTL3 Promotes Osteogenesis by Regulating N6-Methyladenosine-Dependent Primary Processing of hsa-mir-4526

New Mechanism of m6A Methylation Promoting Osteogenic Differentiation of Adipose-Derived Stem Cells Revealed — Study Based on METTL3-Mediated Primary microRNA Splicing Regulation of hsa-mir-4526

1. Academic Background and Research Motivation

Bone tissue engineering has emerged as a cutting-edge interdisciplinary research field due to rapid advances in biotechnology, materials science, and regenerative medicine in recent years. With the growing aging population and the rise in bone defect cases caused by trauma, bone tumors, and other diseases, the need for safe and effective new modalities of bone defect repair and regeneration, especially stem cell-based regenerative strategies, has become an urgent scientific and clinical issue to be addressed.

Among them, human adipose-derived stem cells (hASCs) are widely accepted as ideal seed cells for bone tissue engineering due to their abundant availability, excellent proliferation potential, and differentiation capability. However, the mechanisms underlying osteogenic differentiation of hASCs are complex, regulated by multifaceted molecular networks and epigenetic modifications. These molecular mechanisms remain incompletely elucidated, which constrains the further application of hASCs in bone regenerative medicine and clinical translation.

N6-methyladenosine (m6A) is one of the most prevalent epigenetic modifications in eukaryotic mRNA and a research hotspot in the current RNA field. m6A modification can regulate gene expression and influence life processes such as cell proliferation, apoptosis, and migration, and is believed to be involved in various diseases and stem cell differentiation. The m6A modification process on RNA is reversible and depends on methyltransferase complexes, including key enzymes such as Methyltransferase Like 3 (METTL3). In the context of osteogenic differentiation of stem cells, the regulatory role of m6A modification has garnered increasing attention, but related molecular pathways and targets remain unclear, making it a focal point in academic research.

microRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNAs (about 22nt in length), which function through binding to the 3’ untranslated region of target mRNAs, carrying out post-transcriptional regulation and participating widely in physiological processes such as development, tissue remodeling, and bone metabolism. The maturation of miRNAs proceeds from primary microRNAs (pri-miRNAs) through a series of complex splicing steps involving the Drosha/DGCR8 microprocessor complex. Recent studies have confirmed that m6A modification also participates in the processing and maturation of pri-miRNAs, but the specific mechanisms by which m6A modification mediates miRNA maturation to regulate osteogenic differentiation of stem cells remain unclear.

Based on this, the present study focuses on how METTL3-mediated m6A modification impacts the osteogenic differentiation of hASCs, attempting to reveal new molecular mechanisms in bone regeneration from the perspective of pri-miRNA splicing and maturation, with the goal of providing novel concepts and molecular targets for precise bone defect repair.

2. Source of the Paper and Author Information

The paper is titled “mettl3 promotes osteogenesis by regulating n6-methyladenosine-dependent primary processing of hsa-mir-4526,” authored by Yidan Song, Hongyu Gao, Yihua Pan, Yuxi Gu, Wentian Sun, and Jun Liu (corresponding author). The research team is mainly affiliated with the Department of Orthodontics, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, and West China Longquan Hospital of Sichuan University. This paper was published in 2025 in Stem Cells (DOI: 10.1093/stmcls/sxae089).

3. Research Design and Detailed Workflow (a)

This study systematically reveals a new mechanism for the regulation of hASCs osteogenic differentiation by METTL3/m6A modification, unfolding along the molecular pathway of ‘epigenetic modification—microRNA—osteogenic target gene,’ through a series of in vitro and in vivo experiments, transcriptomic and epitranscriptomic sequencing, and molecular mechanism validation.

1. Main Experimental Workflow and Grouping

• In Vitro Research Workflow

  • hASCs Culture and Osteogenic Induction: hASCs from passages 3–7 were used, divided into undifferentiated (u-hASCs) and osteogenically differentiated groups (d-hASCs). Osteogenic induction uniformly lasted 7 days.
    
  • METTL3 Overexpression and Knockdown: Lentiviral vectors were used to generate METTL3 overexpression, knockdown, and negative control groups.
    
  • Molecular Biological Analyses: Included Western blot for detecting proteins such as METTL3, ALP, RUNX2, TUBB3; immunofluorescence (IF) for osteogenic markers (e.g., RUNX2); as well as alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining to evaluate osteogenic potential.
    
  • miRNA and Target Gene Functional Studies: Agomirs/Antagomirs were used to upregulate/downregulate hsa-mir-4526 expression, with functional rescue experiments performed.
    
  • RNA Methylation and miRNA Splicing Mechanism: MERIP-seq identified pri-miRNAs with differential m6A peaks before and after osteogenic induction; MERIP-qPCR quantified m6A modification on individual miRNAs; RIP and Co-IP explored the interaction between pri-miRNA and DGCR8, METTL3, etc.
    
  • miRNA Target Gene Screening and Validation: Target genes were screened using TargetScan, miRWalk, and in-house transcriptome sequencing, followed by dual-luciferase reporter assays and RIP-qPCR to confirm interaction.
    
  • Functional and Mechanistic Study of Target Gene: siRNA was used to interfere with TUBB3 expression, and its effect on osteogenesis was analyzed via ARS/ALP staining and detection of osteogenic proteins.

• In Vivo Animal Experiments

  • Key Cell Lines Loaded onto GelMA Scaffolds: Four groups (METTL3 intervention/control, hsa-mir-4526 intervention/control) were each transplanted into 4mm calvarial defect models in nude mice.
    
  • After 8 weeks, samples were collected for micro-CT 3D reconstruction, quantitative analysis of bone parameters (BMD/BV/TV/Tb.Sp), and histological evaluations (HE and Masson’s trichrome staining/ IHC) to assess new bone formation and expression of bone proteins.

• Transcriptomic Data and Bioinformatics Analysis

  • RNA-seq was performed on SITUBB3/SINC groups, with FASTP for quality control, DESeq2 for differential gene analysis, and ClusterProfiler for GO/KEGG enrichment; PPI network analysis identified bone metabolism–related key pathways.

2. Core Innovative Methods/Technologies in Detail

• MERIP-seq and MERIP-qPCR: Precisely identify and quantify m6A modification peaks on RNA, capturing epigenetic dynamics during osteogenic differentiation.
• Co-immunoprecipitation and RIP: Reveal protein–RNA/protein–protein interactions to verify that METTL3 promotes pri-miRNA processing via complexing with DGCR8.
• Integrated miRNA Target Screening and Validation: Combined database prediction, algorithms, and empirical data with dual-luciferase and AGO2-RIP to ensure target screening is accurate and functionally relevant.
• In Vivo Scaffold Implantation for Bone Defect Repair: Comprehensive evaluation using 3D CT and histology, enhancing the translational significance of the findings.

4. Main Experimental Results and Logical Process (b)

1. METTL3 Promotes hASCs Osteogenic Differentiation (In Vitro and In Vivo Evidence)

In vitro, the METTL3 knockdown group showed weakened ALP/ARS staining, downregulated ALP/RUNX2 protein expression, and reduced RUNX2-immunofluorescent signals, suggesting impaired osteogenic differentiation. Conversely, METTL3 overexpression enhanced all osteogenesis-associated phenotypes.

In animal models, transplantation of METTL3 knockdown hASCs led to markedly limited calvarial defect repair, 3D reconstruction showed significant reduction of new bone, BMD/BV/TV decreased, Tb.Sp increased, histology and IHC confirmed diminished new bone formation and reduced OCN/ALP osteogenic proteins. Overexpression of METTL3 had the opposite effect, indicating consistent in vivo and in vitro osteo-promotive functions.

2. m6A Modification Promotes pri-miRNA Maturation (Representative: pri-mir-4526)

MERIP-seq revealed dozens of pri-miRNAs with significant alterations in m6A modification levels before and after osteogenic induction (e.g., pri-mir-⁴⁵²⁶⁄₅₁₉₀, pri-mir-6773). Further confirmed by qPCR and MERIP-qPCR, m6A modification on pri-mir-4526 is upregulated during osteogenic differentiation, with corresponding mature miRNA also increased. Interference experiments showed that pri-mir-⁴⁵²⁶⁄₅₁₉₀ displayed a negative correlation with METTL3 expression and its m6A modification, while mature hsa-mir-4526 was positively correlated. RIP/Co-IP/mutation analyses demonstrated that METTL3 enhances binding of pri-mir-4526 to DGCR8 by increasing its m6A modification, facilitating maturation to hsa-mir-4526; mutation of the key A site greatly attenuated this process, directly proving the necessity of m6A modification.

3. hsa-mir-4526 Promotes hASCs Osteogenic Differentiation (Functional Evidence In Vitro and In Vivo)

Downregulation of hsa-mir-4526 led to significantly inhibited osteogenic differentiation, as shown by decreased RUNX2 protein/ARS/ALP staining and quantification in vitro; overexpression enhanced all osteogenic indicators. Importantly, in vivo, new bone formation was significantly higher in the hsa-mir-4526 agomir group compared with controls, while antagomir suppressed new bone generation; bone defect repair capacity, BMD/BV/TV/Tb.Sp, histology, and protein expressions were all in agreement. Rescue experiments indicated that hsa-mir-4526 could strongly restore METTL3 knockdown–induced osteogenesis inhibition, suggesting a coordinated role in the same pathway.

4. Regulation of Downstream Target Gene TUBB3 by METTL3-hsa-mir-4526 Achieves Osteogenic Modulation

TUBB3 (Tubulin beta 3) was screened as the key downstream target of hsa-mir-4526. Triple screening by database, RNA-seq, dual-luciferase reporter assay, and AGO2-RIP confirmed that hsa-mir-4526 directly binds and downregulates TUBB3. During osteogenic differentiation, TUBB3 expression continuously decreased; METTL3/hsa-mir-4526 activation led to further TUBB3 reduction, and their knockdown caused TUBB3 upregulation. TUBB3 interference enhanced RUNX2/ALP and osteogenic staining, indicating TUBB3’s role as a negative regulator. Rescue experiments further confirmed that TUBB3 knockdown could reverse osteogenic inhibition caused by METTL3/hsa-mir-4526 loss, providing molecular evidence for the functional pathway.

5. Molecular Mechanisms of TUBB3 in Osteogenic Regulation (Transcriptomics and Bioinformatics)

RNA-seq analysis of si-TUBB3 group revealed downstream involvement in pathways such as cell metabolism, calcium homeostasis, and osteoclast differentiation. PPI analysis highlighted key bone metabolism gene networks, providing a bioinformatic basis for deeper investigation into the molecular foundation of TUBB3 in osteogenic regulation.

5. Conclusions, Application Value, and Significance ©

For the first time, this study uncovers the molecular pathway wherein METTL3 promotes m6A modification of pri-mir-4526, accelerates its binding to DGCR8 and maturation into hsa-mir-4526, downregulates the negative regulatory gene TUBB3, and ultimately positively regulates the osteogenic differentiation of hASCs. This builds a complete molecular mechanism from epigenetic modification to microRNA, to target gene, and then to phenotype. This discovery not only broadens the understanding of m6A function in stem cell osteogenesis, but also provides key molecular targets and mechanistic bases for bone tissue engineering and clinical bone regeneration.

Specific significance includes:

• Scientific Significance: First clarification of the role of m6A in regulating stem cell fate via pri-miRNA splicing and maturation, filling a theoretical gap in the field.
  
• Methodological Significance: Integration of transcriptomics, epitranscriptomics, and functional animal models, providing an innovative, multi-dimensional research paradigm.
• Applied Significance: Supplies potential targets (such as METTL3/m6A modification, miR-4526, TUBB3) for bone defect repair, bone regeneration, and osteogenesis-related genetic diseases, facilitating development of next-generation gene or epigenetic therapies.

6. Research Highlights and Innovations (d)

• For the first time, the core role and molecular mechanism of m6A modification of pri-mir-4526 in the osteogenic differentiation of adipose-derived stem cells were revealed.
• Complete establishment of the METTL3–pri-mir-4526/hsa-mir-4526–TUBB3 axis as a new regulatory pathway for osteogenesis, providing innovative theoretical support for bone regeneration.
• Use of a new MERIP-seq+MERIP-qPCR platform for high-throughput analysis of epigenetic regulation patterns of miRNAs during osteogenic differentiation.
• Clearly demonstrated, in animal models, the efficacy of these molecular events in real bone defect repair, laying a solid foundation for future translational applications and drug screening based on underlying mechanisms.

7. Supplement and Outlook (e)

This study showcases the cutting-edge advances in epigenetic modification research in China’s oral regenerative medicine field, highlighting high integration of multi-omics, in vivo and in vitro functional validation, and molecular mechanism dissection. In the future, this molecular axis is likely to be prioritized in preclinical research for bone tissue engineering, and offers reference for exploring m6A–microRNA–target gene pathways in other directions of stem cell differentiation. The research methods and pathway models can also be extended to other diseases (such as osteoporosis, impaired fracture healing, etc.). The research team also suggests subsequent development of small molecule screening and targeted delivery platforms for METTL3/m6A-related drugs, to further achieve precision individualized bone repair strategies.

Summary

Integrating molecular epigenetics and bone tissue engineering, this paper innovatively reveals the crucial regulatory pathway of “METTL3 → pri-mir4526 m6A modification and maturation → hsa-mir-4526 → TUBB3 → osteogenic differentiation”, opening new directions for understanding the mechanism and clinical regeneration of bone defects with adipose-derived stem cells. This work not only enriches basic science, but also provides a solid theoretical and practical foundation for future translational medicine and therapeutic interventions for bone defect diseases.