miR-24通过调控GNAI3影响成骨分化的机制研究

Studies demonstrated that GNAI3 gene mutation could cause jaw deformity, and our previous studies found that the low expression of GNAI3 in osteoblasts showed a significantly decreased level of osteogenesis, accompanied by the effects of MAPK signal pathway, moreover, the expressions of 8 miRNAs including miR-24 were significantly downregulated in serum of young people with mandibular protrusion. Bioinformatics analysis reminded that there were possible binding sites between miR-24 and GNAI3 3’UTR, miR-24 may regulate osteogenesis by gene silencing. Therefore, we propose that miR-24 might regulate the MAPK signal pathway and influence the osteogenesis by regulating GNAI3. We will test this hypothesis in cell biological characteristics, molecular biological mechanism and research in vivo. In this study, we investigated the mechanism of miR-24 on GNAI3 in osteoblasts in order to improve the possibility of the application of osteoblasts in the treatment of mandibular protrusion and congenital mandibular defects.

他人研究证明GNAI3基因突变可引起颌骨畸形，我们前期研究发现低表达GNAI3的成骨细胞成骨分化能力显著减弱，并影响MAPK信号通路，而下颌生长过度的青少年患者血清中miR-24等8种miRNA的表达显著下调，生物信息学分析发现miR-24与GNAI3的3’UTR存在结合位点，miR-24可能通过基因沉默作用调控成骨分化。由此我们提出假说：miR-24通过调控GNAI3影响MAPK信号通路进而影响成骨分化。为验证这一假说，本课题拟从分子信号、细胞特性和动物实验三个层面阐明。本研究探索成骨细胞细胞中，miR-24对GNAI3的调控机制，以期提高成骨细胞应用于治疗下颌前突和先天性颌骨缺损的可能性。

背景：他人研究证明GNAI3基因突变可引起颌骨畸形，前期研究证实低表达GNAI3的成骨细胞成骨分化能力显著减弱，而下颌生长过度的青少年患者血清中miR-24-2-5p的表达显著下调，生物信息学分析发现miR-24-2-5p与GNAI3的3’UTR存在结合位点，miR-24-2-5p可能通过基因沉默作用调控成骨分化。.主要研究内容：本研究从分子信号、细胞特性和动物实验三个层面阐明miR-24-2-5p通过Gnai3对成骨细胞及斑马鱼胚胎成骨分化的影响和调控分子机制。.关键数据及重要结果：成骨细胞矿化诱导过程中miR-24-2-5p呈负增长，构建高低表达miR-24-2-5p的成骨细胞模型，检测显示miR-24-2-5p负向调控细胞功能和成骨分化能力；双荧光素酶报告基因检测证实miR-24-2-5p与Gnai3的结合，低表达miR-24-2-5p的成骨细胞中Gnai3的表达升高，提示miR-24-2-5p靶向结合并负向调控Gnai3；构建低表达Gnai3的成骨细胞，检测显示低表达Gnai3负向调控细胞功能和成骨分化作用；低表达miR-24-2-5p的成骨细胞转染Gnai3敲低慢病毒，显示低表达miR-24-2-5p的正向调控作用减弱，其细胞增殖、迁移和成骨分化能力得到部分挽救；斑马鱼胚胎显微注射miR-24-2-5p MO，其胚胎发育无显著差异；显微注射Gnai3 MO，其胚胎体型短小，下颌过小位置后缩，软骨发育和椎骨钙化下降；mir-24-2-5p MO与gnai3 MO共注射，相比于单注射mir-24-2-5p MO，其胚胎体型短小，下颌过小位置后缩，软骨发育和椎骨钙化下降；低表达miR-24-2-5p成骨细胞和低表达Gnai3成骨细胞中P-JNK和P-P38的表达增高，低表达Gnai3成骨细胞中加入p-JNK激活剂和p-P38激活剂，其分化能力增高，证实Gnai3通过MAPK信号通路调控成骨细胞的分化。.科学意义：本研究证实miR-24-2-5p通过调控Gnai3-P-JNK/P-P38信号轴调控成骨细胞分化，为颌面部骨缺损修复和治疗下颌前突提供新思路。