Microcystin-LR prenatal exposure induces coronary heart disease through macrophage polarization imbalance mediated by trophoblast-derived extracellular vesicles.

Trophoblast-derived extracellular vesicles (T-EVs) increase during pregnancy and act as a critical signal in macrophage polarization. However, MC-LR significantly affected the miRNA expression profile of T-EVs. Upon internalization into macrophages, T-EVs-derived miR-377-3p specifically targets the 3'UTR region of NR6A1 to inhibit the gene expression. Silencing of transcription suppressor NR6A1 leads to abnormal activation of the downstream mTOR/S6K1/SREBP pathway, inducing metabolic reprogramming and ultimately leading to M1 polarization of macrophages. Overall design: HTR-8/Svneo cells cultured in exosomes free medium were treated with 0 or 1 µM MC-LR (A-LR-M001, TAIWAN ALGAL SCIENCE INC) for 24 h, after which the cell culture medium was collected for T-EVs extraction by differential centrifugation (300 g/10 min, 2000 g/20 min, 10000 g/30 min and 100000 g/70 min).Then the RNA was extracted for miRNA sequencing

滋养层细胞衍生的细胞外囊泡（Trophoblast-derived extracellular vesicles, T-EVs）在妊娠进程中含量升高，并在巨噬细胞极化过程中发挥关键信号调控作用。然而，微囊藻毒素-LR（MC-LR）可显著改变T-EVs的miRNA表达谱。当被巨噬细胞内吞后，T-EVs来源的miR-377-3p可特异性靶向NR6A1基因的3'非翻译区（3' untranslated region, 3'UTR），从而抑制该基因的表达。沉默转录抑制因子NR6A1会导致下游mTOR/S6K1/SREBP通路异常激活，诱导代谢重编程，最终促使巨噬细胞向M1表型极化。
总体实验设计：将培养于无外囊泡培养基中的HTR-8/Svneo细胞分别用0或1 µM浓度的MC-LR（A-LR-M001，台湾藻类科学公司，TAIWAN ALGAL SCIENCE INC）处理24小时，随后收集细胞培养液，通过差速离心法（300 g/10 min、2000 g/20 min、10000 g/30 min及100000 g/70 min）提取T-EVs。之后提取RNA用于miRNA测序。