[Prickle planar cell polarity protein 1 involved in the pathogenesis of skeletal class Ⅲ malocclusion].

Objective: To explore the mechanisms of prickle planar cell polarity protein 1 (PRICKLE1) involved in the occurrence of skeletal Class Ⅲ malocclusion. Methods: After extracting the genomic DNA of all family members of the skeletal Class Ⅲ malocclusion pedigree with maxillary hypoplasia collected in the Department of Orthodontics at the Affiliated Stomatological Hospital of Nanjing Medical University in October 2021, whole exome sequencing and Sanger sequencing were performed to screen pathogenic genes/mutation sites and validate the mutations. Jaw tissue was collected during the operation of orthognathic patients who were treated in the Department of Oral and Maxillofacial Surgery at the same hospital from October 2021 to December 2022. Following the extraction of human jaw bone marrow mesenchymal stem cells and transfection with overexpressing lentivirus (lentiviruses overexpressing the gene of interest served as the wild group, lentiviruses overexpressing mutation site served as the mutant group) and knockdown lentivirus (divided into knockdown group 1 and 2, with transfection interference negative lentiviruses as the control group). Various assays including real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, proliferation and Transwell assays, alkaline phosphatase staining and alizarin red staining were performed. Construction of zebrafish animal model, morpholino oligonucleotide (MO) were injected to knock down the expression of prickle1a and prickle1b in zebrafish (co-knocking group), and the control group was injected with standardized MO as a reference. Transcriptome sequencing, enrichment analysis and co-expression analysis were performed on the zebrafish craniofacial tissues of the two groups. Results: Two patients of this family carried this mutation PRICKLE1 c.113C>T. The transfection experiments showed that compared with the wild group (relative expression of PRICKLE1 was 21.97±0.60), the relative expression of mutant group (5.05±0.05) was significantly reduced (P<0.05), and cell proliferation and migration ability significantly enhanced (P<0.05), and osteogenic differentiation ability was significantly reduced (P<0.05). Compared with the control group, the proliferation and migration ability of cells in the two knockdown groups were significantly enhanced (P<0.05), and the osteogenic differentiation ability was significantly reduced (P<0.05). Zebrafish model experiments showed the width of the ethmoid plate was significantly reduced in the co-knocking group (282.50±61.77, t=5.29, P<0.001) compared with the control group (338.80±24.92). Transcriptome data and enrichment analysis showed that the differentially expressed genes were significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway after the simultaneous knockdown of prickle1a and prickle1b in zebrafish. Conclusions: PRICKLE1 c.113C>T mutation might suppress the osteoblastic differentiation ability of jaw bone marrow mesenchymal stem cells by downregulating the MAPK signaling pathway, thereby involving the development of skeletal Class Ⅲ malocclusion.目的： 探究刺平面细胞极性蛋白1（prickle planar cell polarity protein 1，PRICKLE1）基因变异参与骨性Ⅲ类错（牙合）畸形发生的分子机制。 方法： 收集2021年10月于南京医科大学附属口腔医院正畸科就诊的骨性Ⅲ类错（牙合）畸形伴上颌骨发育不足家系1个，提取该家系成员基因组DNA进行全外显子组测序，筛选致病基因及突变位点，Sanger测序验证突变序列。收集2021年10月至2022年12月于南京医科大学附属口腔医院口腔颌面外科就诊的正颌患者手术过程中切除的颌骨组织，提取并培养人颌骨骨髓间充质干细胞，转染过表达慢病毒（过表达目的基因的慢病毒为野生组，过表达突变位点的慢病毒为突变组）和敲降慢病毒（分为敲降1组和2组，以干扰阴性慢病毒为对照组），开展实时荧光定量PCR（real-time fluorescence quantitative PCR，RT-qPCR）实验、蛋白质印迹法实验、增殖实验、Transwell实验、碱性磷酸酶和茜素红染色。构建斑马鱼模型，注射吗啉代寡聚核苷酸（morpholino oligonucleotide，MO），在斑马鱼体内敲低prickle1a和prickle1b的表达（共敲组），对照组注射标准化MO作为参照。对2组斑马鱼颅面部组织进行转录组测序、富集分析和共表达分析。 结果： 该家系2例患者均携带PRICKLE1 c.113C>T突变。转染实验显示，相比野生组（PRICKLE1相对表达量为21.97±0.60），突变组人颌骨骨髓间充质干细胞PRICKLE1相对表达量（5.05±0.05）显著降低（P<0.05），细胞增殖能力和迁移能力显著增强（P<0.05），成骨分化能力显著减弱（P<0.05）；相比对照组，2个敲降组细胞的增殖能力和迁移能力均显著增强（P<0.05），成骨分化能力均显著减弱（P<0.05）。斑马鱼模型实验显示，与对照组（338.80±24.92）相比，共敲组斑马鱼筛板宽度显著减小（282.50±61.77）（t=5.29，P<0.001）。转录组测序富集分析结果显示，共敲组和对照组的差异基因在丝裂原活化蛋白激酶信号通路上富集明显。 结论： PRICKLE1 c.113C>T突变下调丝裂原活化蛋白激酶信号通路，抑制颌骨骨髓间充质干细胞的成骨分化能力，进而参与骨性Ⅲ类错（牙合）畸形的发生发展。.