The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, PR China.
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, PR China.
Environ Toxicol Pharmacol. 2020 Nov;80:103438. doi: 10.1016/j.etap.2020.103438. Epub 2020 Jun 20.
Cleft palate is a common congenital maxillofacial malformation in newborns. All-trans retinoic acid (atRA) is an ideal exogenous stimulus to construct a mouse cleft palate model. However, the precise pathogenic mechanism remains to be elucidated. In our study, to explore the toxicity of atRA on palatal shelves during different stages of palate development, a total of 100 mg/kg atRA was administered to C57BL/6 mice at embryonic day 10.5 (E10.5). Mouse embryonic palatal shelves at E13.5, E14.5, E15.5, and E16.5 were collected for RNA extraction and histological treatment. Changes in gene expression were tested through RNA-seq. Selected differentially expressed genes (DEGs) related to metabolic pathways, such as Ptgds, Ttr, Cyp2g1, Ugt2a1 and Mgst3, were validated and analyzed by Quantitative real-time PCR (qRT-PCR). In addition, Gene Oncology analysis showed that transcriptional changes of genes from extracellular matrix (ECM) components, such as Spp1, and crystallin family might play important role in palatal shelves elevation (E13.5-E14.5). Therefore, the protein expression level of Ttr and Spp1 from E13.5 to E16.5 were tested by immunohistochemistry (IHC). Besides, the mRNA level of Spp1, were down-regulated at E16.5 and the protein were down-regulated at E15.5 and E16.5 in all-trans retinoic acid group, suggesting that atRA may involve in palatal bone formation by regulating Spp1. Overall, gene transcriptional profiles were obviously different at each time point of palate development. Thus, this study summarized some pathways and genes that may be related to palatogenesis and cleft palate through RNA-seq, to provide a direction for subsequent studies on the mechanism and targeted therapy of cleft palate.
腭裂是新生儿常见的先天性颌面畸形。全反式视黄酸（atRA）是构建小鼠腭裂模型的理想外源性刺激物。然而，其确切的发病机制仍有待阐明。在我们的研究中，为了探讨 atRA 在腭突发育的不同阶段对腭的毒性作用，在胚胎第 10.5 天（E10.5）向 C57BL/6 小鼠给予 100mg/kg 的 atRA。收集 E13.5、E14.5、E15.5 和 E16.5 日龄的小鼠胚胎腭突进行 RNA 提取和组织学处理。通过 RNA-seq 检测基因表达的变化。选择与代谢途径相关的差异表达基因（DEGs），如 Ptgds、Ttr、Cyp2g1、Ugt2a1 和 Mgst3，通过定量实时 PCR（qRT-PCR）进行验证和分析。此外，基因肿瘤学分析表明，细胞外基质（ECM）成分相关基因如 Spp1 和晶体蛋白家族的转录变化可能在腭突升高（E13.5-E14.5）中发挥重要作用。因此，通过免疫组织化学（IHC）检测 E13.5 至 E16.5 时 Ttr 和 Spp1 的蛋白表达水平。此外，在全反式视黄酸组中，Spp1 的 mRNA 水平在 E16.5 时下调，蛋白水平在 E15.5 和 E16.5 时下调，提示 atRA 可能通过调节 Spp1 参与腭骨形成。总之，腭发育的各个时间点的基因转录谱明显不同。因此，本研究通过 RNA-seq 总结了一些可能与腭发生和腭裂相关的途径和基因，为腭裂发生机制和靶向治疗的后续研究提供了方向。
