FTO-mediated m6A modification regulates the osteogenic differentiation of ADSCs by targeting FOXO1

Abstract

Using adipose-derived stem cells (ADSCs) has recently become a crucial approach for treating bone defects owing to their ease of accessibility and substantial differentiation potential. N6-methyladenosine (m6A) modification greatly influences biological processes and determines the differentiation fate of stem cells. However, the specific mechanisms by which m6A modification influences the osteogenic differentiation of ADSCs remain unclear. We identified FOXO1 as the key m6A-modified gene during the osteogenesis of ADSCs. Furthermore, demethylase FTO enhanced RUNX2 expression while inhibiting PPARG expression by modifying FOXO1, thereby facilitating ADSC osteogenesis. FTO knockdown inhibited ADSC migration and proliferation and impaired osteogenesis by suppressing FOXO1. At the mechanistic level, we first revealed that FTO was exported to the cytoplasm and then directly bound with FOXO1 mRNA at its 1760th bp site. Consistent use of non-steroidal anti-inflammatory drugs (NSAIDs) containing FTO inhibitors impeded ADSC-mediated bone formation both in vivo and in vitro. In summary, our study reveals the role of m6A modification based on the FTO–FOXO1–RUNX2/PPARG axis in regulating the osteogenic differentiation of ADSCs, thereby improving the clinical use of ADSCs and providing strategies for related drug applications in bone regeneration.

1. Introduction

Bone defects are caused by trauma, tumors, infections, congenital deformities and the exacerbation of related diseases. Over 6 million patients suffer from bone defects annually in China [1]. Owing to the limited regenerative capacity of bone tissue, defects over the critical size often result in delayed or even nonunion fractures. Traditional transplantation material, including autogenous, allograft, and synthetic bone substitutes, exhibits certain limitations, such as donor site defects, poor bone quality, and confined applicability of graft materials. The latest bone tissue engineering concept combines biomaterials with cells or healing factors to promote bone regeneration [2]. Stem cell therapy, an important branch of regenerative medicine, provides a new option for patients who are unfit for traditional treatments.

Mesenchymal stem cells (MSCs) are a common type of somatic stem cells known for their self-renewal and differentiation abilities. They are primarily derived from bone marrow, adipose tissue, and umbilical cord [3]. Compared with bone marrow mesenchymal stem cells (BMSCs), ADSCs are better suited for bone tissue regeneration. Bone marrow aspiration is challenging, and BMSCs are uncommon, constituting less than 0.01% of monocytes [4]. Compared with BMSCs, ADSCs are more readily available and abundant. Additionally, ADSCs demonstrate (1) similar or even higher regenerative capacity; (2) higher proliferative activity; (3) greater secreted regeneration factors; (4) immunosuppressive ability [5]. To stimulate angiogenesis and osteogenesis, ADSCs release a variety of peptides, hormones, and growth factors. They also produce chemokines to recruit endogenous stem cells to bone defect sites [6].

Epigenetic regulation of gene expression is critical to cellular function, as it controls the synthesis of proteins and modulatory molecules. In eukaryotes, DNA is tightly compacted into chromatin, limiting transcriptional machinery access. However, epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNAs, can induce heritable changes in gene expression by altering chromatin structure and accessibility [7]. These processes extensively influence cell phenotype and function, which is crucial for MSC fate determination, differentiation, and functional specialization, with broad implications for stem cell biology and regenerative medicine [8]. Over 80% of all RNA methylation occurs ...