Circular RNA circ-ARFIP2 regulates proliferation, migration and invasion in human vascular smooth muscle cells via miR-338-3p-dependent modulation of KDR

Dysfunction of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of intracranial aneurysm (IA). Circular RNAs (circRNAs) have been implicated in the pathogenesis of IA by reducing MicroRNA (miRNA) activity. In this paper, we investigated the precise roles of circRNA ADP ribosylation factor interacting protein 2 (circ-ARFIP2, circ_0021001) in VSMC dysfunction. The levels of circ-ARFIP2, miR-338-3p and kinase insert domain receptor (VEGFR2/KDR/Flk-1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R and subcellular fractionation assays were used to assess the stability and localization of circ-ARFIP2, respectively. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell invasion was measured by transwell assay. Cell proliferation was gauged by 5-Ethynyl-2'-Deoxyuridine (EdU) assay. Cell migration was evaluated by transwell and wound-healing assays. Targeted correlations among circ-ARFIP2, miR-338-3p and VEGFR2/KDR/Flk-1 were validated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ-ARFIP2 and VEGFR2/KDR/Flk-1 were underexpressed and miR-338-3p was overexpressed in the arterial wall tissues of IA patients. Overexpression of circ-ARFIP2 in human umbilical artery smooth muscle cells (HUASMCs) showed a significant promotion in cell proliferation, migration and invasion. Mechanistically, circ-ARFIP2 targeted miR-338-3p, and circ-ARFIP2 regulated cell behaviors by miR-338-3p. VEGFR2/KDR/Flk-1 was a direct and functional target of miR-338-3p. Moreover, VEGFR2/KDR/Flk-1 was a downstream effector of circ-ARFIP2 function. Circ-ARFIP2 regulated VEGFR2/KDR/Flk-1 expression by targeting miR-338-3p. Our present findings demonstrated that the increased level of circ-ARFIP2 enhanced HUASMC proliferation, migration and invasion at least in part by the miR-338-3p/VEGFR2/KDR/Flk-1 axis.