Feng Weijia, Chen Ting
Department of Pediatric Orthopaedic, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Yangpu District, Shanghai, 200092, China.
Funct Integr Genomics. 2025 Jan 6;25(1):7. doi: 10.1007/s10142-024-01516-7.
This study investigates the mechanism of PHF20 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). BMSCs from Balb/c mouse were cultured and identified through osteogenesis, adipogenesis, and flow cytometry. After osteogenic induction, the levels of OPN and OCN in BMSCs were detected by RT-qPCR. Alizarin red staining and alkaline phosphatase (ALP) staining were used to evaluate the osteogenic differentiation ability of BMSCs. PHF20, METTL14, and HOXA13 expressions were detected by RT-qPCR and Western blot. After quantitative analysis of m6A level, RNA immunoprecipitation (RIP) was performed to measure the enrichment of IGF2BP3 or m6A on HOXA13 mRNA. HOXA13 mRNA stability was assessed after actinomycin D treatment. PHF20, METT14, and HOXA13 expressions gradually increased during osteogenic differentiation of BMSCs. Suppression of PHF20 expression repressed the osteogenic differentiation of BMSCs, mainly resulted in a decrease in OPN and OCN levels, reduced mineralization, and weakened ALP activity. Mechanistically, PHF20 elevated METTL14 expression by enhancing the enrichment of H3K4me3 on its promoter, and METTL14 strengthened HOXA13 m6A methylation to maintain HOXA13 mRNA stability through IGF2BP3. In conclusion, PHF20 elevates METTL14 expression by enhancing H3K4me3 enrichment on its promoter and enhances HOXA13 mRNA stability via IGF2BP3-mediated m6A modification, thus facilitating HOXA13 expression and eventually inducing osteogenic differentiation of BMSCs.
本研究探讨PHF20在骨髓间充质干细胞（BMSCs）成骨分化中的作用机制。培养来自Balb/c小鼠的BMSCs，并通过成骨、成脂及流式细胞术进行鉴定。成骨诱导后，采用RT-qPCR检测BMSCs中骨桥蛋白（OPN）和骨钙素（OCN）的水平。茜素红染色和碱性磷酸酶（ALP）染色用于评估BMSCs的成骨分化能力。采用RT-qPCR和蛋白质免疫印迹法检测PHF20、METTL14和HOXA13的表达。在对m6A水平进行定量分析后，进行RNA免疫沉淀（RIP）实验以检测IGF2BP3或m6A在HOXA13 mRNA上的富集情况。用放线菌素D处理后评估HOXA13 mRNA的稳定性。在BMSCs成骨分化过程中，PHF20、METT14和HOXA13的表达逐渐增加。抑制PHF20的表达可抑制BMSCs的成骨分化，主要表现为OPN和OCN水平降低、矿化减少及ALP活性减弱。机制上，PHF20通过增强H3K4me3在其启动子上的富集来提高METTL14的表达，而METTL14通过IGF2BP3增强HOXA13的m6A甲基化以维持HOXA13 mRNA的稳定性。总之，PHF20通过增强H3K4me3在其启动子上的富集来提高METTL14的表达，并通过IGF2BP3介导的m6A修饰增强HOXA13 mRNA的稳定性，从而促进HOXA13的表达并最终诱导BMSCs的成骨分化。
