Department of Plastic Surgery and Burn Center, Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, China.
Department of Medical Imaging, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, China.
Exp Cell Res. 2020 Jan 15;386(2):111734. doi: 10.1016/j.yexcr.2019.111734. Epub 2019 Nov 23.
The molecular mechanisms of abnormal palatogenesis were investigated in this study. A key regulator, miR-106a-5p, and its target pathway were analyzed.
This research is trying to clarify the underlying mechanism of the modulation of miRNA transcription during the formation of cleft palate by 7T and 9.4T NMR metabolomic platforms.
Differentially expressed miRNAs and mRNAs were analyzed by microarray analysis and verified by qRT-PCR. The protein expression in TGFβ signaling pathways were analyzed by Western Blotting. The relationship between miR-106a-5p and TGFβ were analyzed by luciferase reporter assay. Cell apoptosis were analyzed by flow cytometer. And finally, the metabonomics were analyzed by NMR and multivariate data analysis models (MVDA).
The expression of miR-106a-5p increased in cleft palatal tissue and negatively correlated with the protein level of Tgfbr2. The luciferase assay further proved that the tgfbr2 was a direct target of miR-106a-5p. In another aspect, miR-106a-5p increased apoptosis level in palatal mesenchymal cells, possibly because its inhibition of TGFβ signaling pathway. Moreover, low cholesterol and choline levels with high citric acid and lipid levels were observed by 7T and 9.4T NMR metabonomic analysis, which inferred the disorder of cell membrane synthesis in cleft palate formation. Furthermore, transformation from choline to phosphatidylcholine regulated by miR-106a-5p was also disrupted, resulting in phosphatidic choline synthesis disorder and reduced cell membrane synthesis.
The regulatory mechanism of cleft palate was studied at transcriptional and metabolomics levels, which may provide important information in understanding the primary cause of this abnormality.
本研究旨在探讨腭裂发生的分子机制，分析关键调控因子 miR-106a-5p 及其靶通路。
本研究拟利用 7T 和 9.4T NMR 代谢组学平台阐明 miRNA 转录调控在腭裂形成中的潜在机制。
采用微阵列分析和 qRT-PCR 验证差异表达的 miRNA 和 mRNA。通过 Western Blotting 分析 TGFβ 信号通路中的蛋白表达。通过荧光素酶报告实验分析 miR-106a-5p 与 TGFβ 的关系。采用流式细胞术分析细胞凋亡。最后，通过 NMR 和多元数据分析模型（MVDA）进行代谢组学分析。
miR-106a-5p 在腭裂组织中表达增加，与 Tgfbr2 蛋白水平呈负相关。荧光素酶实验进一步证实 tgfbr2 是 miR-106a-5p 的直接靶基因。另一方面，miR-106a-5p 增加了腭中胚层细胞的凋亡水平，可能是通过抑制 TGFβ 信号通路。此外，通过 7T 和 9.4T NMR 代谢组学分析发现，胆固醇和胆碱水平降低，柠檬酸和脂质水平升高，提示腭裂形成过程中细胞膜合成紊乱。此外，miR-106a-5p 调控的胆碱向磷脂酰胆碱的转化也被打乱，导致磷酸胆碱合成紊乱和细胞膜合成减少。
本研究从转录组学和代谢组学水平研究了腭裂的调控机制，为深入了解这种异常的主要原因提供了重要信息。
