Li Yifan, Jiang Chengxian, Ma Teng, Huang Mingwei, Jin Chengri
Department of Stomatology, Affiliated Hospital of Yanbian University(Yanbian Hospital), Yanji, Jilin 133000, China.
Department of Stomatology, Beijing Huairou Hospital, Beijing, 101400, China.
J Craniomaxillofac Surg. 2025 Sep;53(9):1463-1470. doi: 10.1016/j.jcms.2025.06.006. Epub 2025 Jun 23.
This study aimed to investigate the role of the Midline1 gene in secondary palate development by analyzing its expression and function in palatal shelf fusion and morphology.
Initially, twenty mouse embryos were collected for each of the embryonic stages E13.5, E13.75, and E14.5. Whole-mount in situ hybridization was performed approximately ten times to optimize the experimental protocol and to analyze the expression pattern of Midline1 (MID1) in the palatal tissues at these developmental stages. Subsequently, palatal tissues from E13.5 embryos were treated with varying concentrations of Midline1 small interfering RNA (MID1 siRNA), and the knockdown efficiency was evaluated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR), with each concentration tested in triplicate. Based on the results, the most effective concentration, 100 nM MID1 siRNA, was selected for further experiments. Subsequently, twelve E13.5 palatal explants were allocated into two groups: six explants were treated with 100 nM MID1 siRNA (experimental group), and six with scrambled small interfering RNA(Scramble siRNA; control group). After 48 h of in vitro culture, hematoxylin and eosin (HE) staining was performed to evaluate the morphology of palatal shelf fusion. To evaluate apoptotic activity, Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining was performed on both experimental and control groups. Finally, immunohistochemistry and Western blot analyses were conducted to examine the expression levels of Matrix Metalloproteinase 8 (MMP8) and Snail Family Transcription Factors (Snail) proteins in three biological replicates from each group.
Midline1 deficiency resulted in incomplete palatal shelf fusion and significantly reduced apoptosis. Additionally, the knockdown of Midline1 led to the upregulation of Snail and MMP8 gene expression, indicating that Midline1 plays a critical role in regulating epithelial-to-mesenchymal transition and maintaining cytoskeletal stability during palate development.
Midline1 is essential for normal secondary palate development. Its dysregulation disrupts palatal shelf fusion and morphology, potentially contributing to craniofacial abnormalities such as cleft palate. These findings provide new insights into the molecular mechanisms underlying palate development and suggest that Midline1 could be a therapeutic target for addressing cleft palate and related defects.
本研究旨在通过分析中线1基因（Midline1）在腭突融合及形态形成中的表达和功能，探讨其在继发腭发育中的作用。
最初，收集E13.5、E13.75和E14.5三个胚胎阶段的小鼠胚胎各20个。进行了约十次全胚胎原位杂交，以优化实验方案并分析Midline1（MID1）在这些发育阶段腭组织中的表达模式。随后，用不同浓度的Midline1小干扰RNA（MID1 siRNA）处理E13.5胚胎的腭组织，并使用逆转录定量聚合酶链反应（RT-qPCR）评估敲低效率，每个浓度重复测试三次。根据结果，选择最有效的浓度100 nM MID1 siRNA进行进一步实验。随后，将12个E13.5腭外植体分为两组：六个外植体用 100 nM MID1 siRNA处理（实验组），六个用乱序小干扰RNA（Scramble siRNA；对照组）处理。体外培养48小时后，进行苏木精-伊红（HE）染色以评估腭突融合的形态。为评估凋亡活性，对实验组和对照组均进行了末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色。最后，进行免疫组织化学和蛋白质印迹分析，以检测每组三个生物学重复样本中基质金属蛋白酶8（MMP8）和蜗牛家族转录因子（Snail）蛋白的表达水平。
Midline1基因缺失导致腭突融合不完全，凋亡显著减少。此外，Midline1基因敲低导致Snail和MMP8基因表达上调，表明Midline1在腭发育过程中调节上皮-间充质转化和维持细胞骨架稳定性方面起关键作用。
Midline1对正常继发腭发育至关重要。其失调会破坏腭突融合和形态，可能导致诸如腭裂等颅面畸形。这些发现为腭发育的分子机制提供了新见解，并表明Midline1可能是解决腭裂及相关缺陷的治疗靶点。
