This study aimed to determine the metabolites associated with ventricular septal defect (VSD) and the underlying mechanisms. Blood samples and thymus tissues were collected from VSD patients to perform LC-MS-based metabolomics assay and generate iPS cell-derived cardiomyocytes, respectively. VSD rat model was used in vivo study. RT-PCR, western blotting, immunohistochemistry, luciferase activity assay, GFP-LC3 adenovirus and GFP and RFP tfLC3 assay, and transmission electron microscopy were performed to investigate the underlying mechanisms. The metabolites uric acid (UA) and sphingomyelin (SM) increased in the serum of VSD patients, along with enhanced autophagy. The combination of UA and SM treatment could promote autophagy and inhibit EGFR and AKT3 expressions. Overexpression of EGFR and AKT3 suppressed autophagy in UA and SM-treated cardiomyocytes, respectively. Also, lncRNA MEG3 knockdown and overexpression could enhance and inhibit autophagy in UA and SM-treated cardiomyocytes, respectively, through targeting miR-7-5p. Moreover, miR-7-5p mimics and inhibitors promoted and inhibited autophagy in UA and SM-treated cardiomyocytes, respectively, via target EGFR. In VSD rat model, upregulation of MEG3 could reverse high level of autophagy and decrease serum UA and SM. In conclusion, UA and SM are essential VSD-associated metabolic biomarkers and MEG3/miR-7-5p/EGFR axis is critical to the regulation of autophagy in cardiomyocytes.

中文翻译：

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尿酸和鞘磷脂通过lncRNA MEG3 / miR-7-5p / EGFR轴增强iPS细胞起源的心肌细胞的自噬。

这项研究旨在确定与室间隔缺损（VSD）相关的代谢产物及其潜在机制。从VSD患者中采集血液样本和胸腺组织，以进行基于LC-MS的代谢组学分析，并分别生成iPS细胞衍生的心肌细胞。VSD大鼠模型用于体内研究。进行了RT-PCR，蛋白质印迹，免疫组织化学，荧光素酶活性测定，GFP-LC3腺病毒以及GFP和RFP tfLC3测定，以及透射电镜，以研究其潜在机制。VSD患者血清中的代谢产物尿酸（UA）和鞘磷脂（SM）增加，并且自噬增强。UA和SM联合治疗可促进自噬并抑制EGFR和AKT3表达。EGFR和AKT3的过表达分别抑制了UA和SM治疗的心肌细胞的自噬。同样，通过靶向miR-7-5p，lncRNA MEG3的敲低和过表达可以分别增强和抑制UA和SM处理的心肌细胞的自噬。此外，miR-7-5p模拟物和抑制剂分别通过靶EGFR促进和抑制UA和SM处理的心肌细胞的自噬。在VSD大鼠模型中，MEG3的上调可逆转高水平的自噬并降低血清UA和SM。总之，UA和SM是与VSD相关的必不可少的代谢生物标记物，MEG3 / miR-7-5p / EGFR轴对于调节心肌细胞自噬至关重要。此外，miR-7-5p模拟物和抑制剂分别通过靶EGFR促进和抑制UA和SM处理的心肌细胞的自噬。在VSD大鼠模型中，MEG3的上调可逆转高水平的自噬并降低血清UA和SM。总之，UA和SM是与VSD相关的必不可少的代谢生物标记物，MEG3 / miR-7-5p / EGFR轴对于调节心肌细胞自噬至关重要。此外，miR-7-5p模拟物和抑制剂分别通过靶EGFR促进和抑制UA和SM处理的心肌细胞的自噬。在VSD大鼠模型中，MEG3的上调可以逆转高水平的自噬并降低血清UA和SM。总之，UA和SM是与VSD相关的必不可少的代谢生物标记物，MEG3 / miR-7-5p / EGFR轴对于调节心肌细胞自噬至关重要。