Surgical Department of Cardiothoracic Macrovascular, Jingzhou Hospital Affiliated to Yangtze University, No.26 Chuyuan Avenue, Jingzhou District, Jingzhou, 434020, Hubei, China.
Cardiovasc Toxicol. 2024 Feb;24(2):111-121. doi: 10.1007/s12012-024-09833-w. Epub 2024 Feb 20.
Circular RNA (circRNAs) has been confirmed to participate in atherosclerosis (AS) progression. However, the role and mechanism of hsa_circ_0032389 in AS process still need to be further revealed. This study evaluates the role and mechanism of hsa_circ_0032389 in AS process. Platelet-derived growth factor-BB (PDGF-BB) was used to induce human aortic vascular smooth muscle cells (HA-VSMCs). The expression levels of hsa_circ_0032389, microRNA (miR)-513a-5p, and fibroblast growth factor receptor substrate 2 (FRS2) were examined by quantitative real-time PCR. Cell proliferation and migration were analyzed using cell counting kit 8 assay, flow cytometry, EdU assay, transwell assay, and wound healing assay. Protein expression was assessed using western blot analysis. Dual-luciferase reporter and RIP assays were used to confirm RNA interaction. Hsa_circ_0032389 was overexpressed in PDGF-BB-induced HA-VSMCs, and its downregulation inhibited HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate under PDGF-BB treatment. The luciferase activity of hsa_circ_0032389 could be reduced by miR-513a-5p mimic, and both hsa_circ_0032389 and miR-513a-5p were enriched in anti-Ago2, confirming that miR-513a-5p could be sponged by hsa_circ_0032389. MiR-513a-5p inhibitor reversed the effect of hsa_circ_0032389 knockdown on PDGF-BB-induced HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate. Moreover, the luciferase activity of FRS2 was reduced by miR-513a-5p mimic, and both FRS2 and miR-513a-5p were enriched in anti-Ago2, verifying that FRS2 was targeted by miR-513a-5p. MiR-513a-5p suppressed PDGF-BB-induced HA-VSMC viability, cell cycle, EdU positive cell rate, migratory cell number, and wound closure rate by targeting FRS2. Our results indicated that hsa_circ_0032389 enhanced PDGF-BB-induced HA-VSMC proliferation and migration via regulating miR-513a-5p/FRS2 axis.
环状 RNA（circRNAs）已被证实参与动脉粥样硬化（AS）的进展。然而，hsa_circ_0032389 在 AS 过程中的作用和机制仍需要进一步揭示。本研究评估了 hsa_circ_0032389 在 AS 过程中的作用和机制。使用血小板衍生生长因子-BB（PDGF-BB）诱导人主动脉血管平滑肌细胞（HA-VSMCs）。通过实时定量 PCR 检测 hsa_circ_0032389、微小 RNA（miR）-513a-5p 和成纤维细胞生长因子受体底物 2（FRS2）的表达水平。使用细胞计数试剂盒 8 测定细胞增殖和迁移，使用流式细胞术、EdU 测定、Transwell 测定和划痕愈合测定分析细胞迁移，使用 Western blot 分析测定蛋白表达。使用双荧光素酶报告和 RIP 测定来确认 RNA 相互作用。在 PDGF-BB 诱导的 HA-VSMCs 中过表达 hsa_circ_0032389，其下调抑制 PDGF-BB 处理下 HA-VSMC 的活力、细胞周期、EdU 阳性细胞率、迁移细胞数和伤口闭合率。hsa_circ_0032389 的荧光素酶活性可被 miR-513a-5p 模拟物降低，hsa_circ_0032389 和 miR-513a-5p 均富集在抗 Ago2 中，证实 miR-513a-5p 可以被 hsa_circ_0032389 海绵化。miR-513a-5p 抑制剂逆转了 hsa_circ_0032389 敲低对 PDGF-BB 诱导的 HA-VSMC 活力、细胞周期、EdU 阳性细胞率、迁移细胞数和伤口闭合率的影响。此外，FRS2 的荧光素酶活性可被 miR-513a-5p 模拟物降低，并且 FRS2 和 miR-513a-5p 都富集在抗 Ago2 中，验证了 FRS2 是 miR-513a-5p 的靶标。miR-513a-5p 通过靶向 FRS2 抑制 PDGF-BB 诱导的 HA-VSMC 活力、细胞周期、EdU 阳性细胞率、迁移细胞数和伤口闭合率。我们的结果表明，hsa_circ_0032389 通过调节 miR-513a-5p/FRS2 轴增强 PDGF-BB 诱导的 HA-VSMC 增殖和迁移。
