Background: 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.
Methods: 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.
Results: 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.
Conclusions: Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis.
Keywords: Atherosclerosis; Endothelial cell; Hsa_circ_0003204; MiR-370-3p; TGFβR2.