METTL3 mediated m6A methylation to promote mandibular condyle fracture healing via TGFB/Smad signaling pathway

A mandibular condyle fracture rat model was established. Micro-CT scans and biochemical indicators were measured at 1 and 5 weeks post-fracture. The expression levels of m6A methylation-related enzymes were analyzed. METTL3 knockdown and overexpression models were constructed to evaluate structural healing via micro-CT, biomechanical properties, osteogenesis/chondrogenesis-related proteins, and the TGFB/Smad signaling pathway. BMSCs were isolated for in vitro analyses, including cell viability, apoptosis, osteogenic differentiation, signaling pathway protein expression, and MeRIP-seq analysis. MeRIP-qPCR, RNA pull-down, and mRNA stability assays were used to verify METTL3-mediated m6A methylation targets.

本研究构建了下颌髁突骨折大鼠模型。于骨折后1周及5周开展显微CT（micro-CT）扫描并检测生化指标，分析m6A甲基化相关酶的表达水平。构建METTL3敲低与过表达模型，通过显微CT、生物力学特性、成骨/成软骨相关蛋白及转化生长因子β/Smad（TGFB/Smad）信号通路评估骨折结构愈合情况。分离骨髓间充质干细胞（BMSCs）进行体外分析，检测内容包括细胞活力、细胞凋亡、成骨分化、信号通路蛋白表达及甲基化RNA免疫沉淀测序（MeRIP-seq）分析。采用甲基化RNA免疫沉淀定量PCR（MeRIP-qPCR）、RNA下拉实验及mRNA稳定性实验验证METTL3介导的m6A甲基化靶标。