FOXO1转录激活Kcnq1ot1调控BMP4介导LIPUS促进多孔钛合金成骨的分子机制

Porous scaffolds combined with low-intensity pulsed ultrasound (LIPUS) is widely concerned for the possibility of rapid restoration of large-area jaw defects. Our previous studies showed that LIPUS promoted osteogenesis of porous titanium alloys. However, its regulatory mechanism remains unclear. On the basis of RNA-seq analysis, we found that the expression of both Kcnq1ot1 and BMP4 were upregulated, and miR-1187 targeted binding to them was downregulated, which had potential ceRNA regulatory relationship. Besides, we found that FOXO1 could target the promoter region of Kcnq1ot1 and positively regulate the expression of Kcnq1ot1. Therefore, we hypothesized that LIPUS could promote the osteogenesis of porous titanium alloys through ceRNA pathway of Kcnq1ot1/miR-1187/BMP4 transcriptionally activated by FOXO1.To provide theoretical guidance for the application of porous scaffolds combined with LIPUS in the clinical treatment of large-area jaw defects,we will elucidate the mechanism in osteogenesis of porous titanium alloys promoted by LIPUS through ceRNA pathway of Kcnq1ot1/miR-1187/BMP4 transcriptionally activated by FOXO1 using overexpression/silence technology,cell model in vitro and nude-mouse model in vivo.

多孔材料联合低强度脉冲超声波（LIPUS）因有望快速修复大面积骨缺损而引起广泛关注。申请人前期研究发现LIPUS可以促进多孔钛合金成骨作用，但其调控机制不明。在RNA-seq分析基础上，我们发现：LIPUS作用下Kcnq1ot1和BMP4上调，与二者靶向结合的miR-1187下调，具有潜在ceRNA调控关系；同时Kcnq1ot1启动子区与FOXO1有结合位点，FOXO1对Kcnq1ot1有正向调控作用。推测FOXO1通过转录激活Kcnq1ot1/miR-1187/BMP4的ceRNA通路介导LIPUS促进多孔钛合金成骨作用。本项目拟利用过表达/沉默技术、体外细胞模型和体内裸鼠模型方法，阐明LIPUS作用下FOXO1转录激活Kcnq1ot1/miR-1187/BMP4构成的ceRNA通路调控多孔钛合金成骨作用的机制，为多孔材料联合LIPUS应用于大面积颌骨缺损的临床治疗提供理论指导。

多孔材料联合低强度脉冲超声波（LIPUS）有望成为快速修复大面积颌骨缺损的新方法，但LIPUS促进多孔材料成骨的分子生物学机制尚不清楚。既往研究发现LIPUS可促进多孔钛合金材料成骨，且加载及未加载LIPUS的支架材料上成骨细胞全转录组测序分析、生物信息学预测分析、预实验结果显示：1.Mmu-miR-1187在LIPUS刺激后表达显著下调， BMP4为miR-1187的靶基因，BMP4是高成骨活性的重要生长因子。2.与人同源性较高的lncGm29630在LIPUS作用下显著上调，miR-1249-5p可同时靶向性结合lncGm29630和Wnt2b，过表达lncGm29630可激活Wnt/β-Catenin信号通路。3.LIPUS作用下mmu_CIRCpedia_9393显著上调，mmu_CIRCpedia_9393为miR-217-5p、miR-326-3p的海绵，SIRT1为miR-217-5p、miR-326-3p的共同的靶基因，mmu_CIRCpedia_9393的高表达可促进MC3T3-E1成骨分化。4.LIPUS可抑制破骨细胞分化，m6A修饰水平在破骨细胞分化中明显上升，去甲基化酶FTO在M6A修饰中发挥重要作用。因而本课题分别从成骨细胞miRNA、ceRNA机制、circRNA、破骨细胞RNA修饰角度出发，分别建立体外成骨细胞与多孔钛合金支架材料复合培养、破骨细胞培养、体内大鼠下颌骨缺损模型，通过多种方法检测LIPUS作用下，上述指标表达水平变化及其对成骨分化、骨长入、破骨分化的影响及其相互调控作用机制。研究结果表明：1.LIPUS可通过抑制miR-1187上调BMP4进一步促进多孔钛合金材料成骨。2.LIPUS可通过激活lncGm29630/miR-1249-5p/Wnt2b轴促进多孔钛合金支架材料内细胞成骨分化。3.LIPUS可通过激活mmu_CIRCpedia_9393/miR-217-5p、miR-326-3p/SIRT1轴促进多孔钛合金支架内成骨。4.低强度脉冲超声波可通过上调FTO表达调控RNA甲基化修饰抑制破骨细胞分化。上述研究结果探索了LIPUS促进多孔钛合金成骨、抑制破骨细胞分化的分子生物学机制，为“细胞因子联合多孔材料及LIPUS加载”应用于大面积骨缺损的临床治疗方案提供理论指导。