Circular RNAs (circRNAs) represent a class of non-coding RNAs (ncRNAs) which are widely expressed in mammals and tissue-specific, of which some could act as critical regulators in the atherogenesis of cerebrovascular disease. However, the underlying mechanisms by which circRNA regulates the ectopic phenotype of endothelial cells (ECs) in atherosclerosis remain largely elusive. CCK-8, transwell, wound healing and Matrigel assays were used to assess cell viability, migration and tube formation. QRT-qPCR and Immunoblotting were used to examine targeted gene expression in different groups. The binding sites of miR-370-3p (miR-370) with TGFβR2 or hsa_circ_0003204 (circ_0003204) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The localization of circ_0003204 and miR-370 in ECs were investigated by fluorescence in situ hybridization (FISH). Gene function and pathways were enriched through Metascape and gene set enrichment analysis (GSEA). The association of circ_0003204 and miR-370 in extracellular vesicles (EVs) with clinical characteristics of patients were investigated using multiple statistical analysis. Circ_0003204, mainly located in the cytoplasm of human aorta endothelial cells (HAECs), was upregulated in the ox-LDL-induced HAECs. Functionally, the ectopic expression of circ_0003204 inhibited proliferation, migration and tube formation of HAECs exposed to ox-LDL. Mechanically, circ_0003204 could promote protein expression of TGFβR2 and its downstream phosph-SMAD3 through sponging miR-370, and miR-370 targeted the 3′ untranslated region (UTR) of TGFβR2. Furthermore, the expression of circ_0003204 in plasma EVs was upregulated in the patients with cerebral atherosclerosis, and represented a potential biomarker for diangnosis and prognosis of cerebrovascular atherogenesis. Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis.

中文翻译：

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环状RNA circ_0003204通过miR-370-3p/TGFβR2/phosph-SMAD3轴抑制动脉粥样硬化内皮细胞增殖、迁移和管形成

环状RNA（circRNA）代表一类非编码RNA（ncRNA），它们在哺乳动物中广泛表达并具有组织特异性，其中一些可以作为脑血管疾病动脉粥样硬化形成的关键调节因子。然而，circRNA 调节动脉粥样硬化中内皮细胞（ECs）异位表型的潜在机制在很大程度上仍然难以捉摸。CCK-8、transwell、伤口愈合和基质胶测定用于评估细胞活力、迁移和管形成。QRT-qPCR 和免疫印迹用于检查不同组中的目标基因表达。使用一系列生物信息学工具预测 miR-370-3p (miR-370) 与 TGFβR2 或 hsa_circ_0003204 (circ_0003204) 的结合位点，并使用双荧光素酶测定和 RNA 免疫沉淀 (RIP) 测定进行验证。通过荧光原位杂交（FISH）研究了 circ_0003204 和 miR-370 在 ECs 中的定位。通过 Metascape 和基因集富集分析 (GSEA) 富集了基因功能和通路。使用多重统计分析研究了细胞外囊泡 (EV) 中 circ_0003204 和 miR-370 与患者临床特征的关联。Circ_0003204 主要位于人主动脉内皮细胞 (HAECs) 的细胞质中，在 ox-LDL 诱导的 HAECs 中上调。在功能上，circ_0003204 的异位表达抑制了暴露于 ox-LDL 的 HAEC 的增殖、迁移和管形成。机械上，circ_0003204 可以通过海绵 miR-370 促进 TGFβR2 及其下游 phosph-SMAD3 的蛋白质表达，而 miR-370 靶向 TGFβR2 的 3' 非翻译区（UTR）。此外，circ_0003204在脑动脉粥样硬化患者血浆EVs中的表达上调，代表了脑血管粥样硬化诊断和预后的潜在生物标志物。Circ_0003204 可以作为动脉粥样硬化中异位内皮失活的新型刺激剂和脑动脉粥样硬化的潜在生物标志物。