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Hsa_circ_0032389 Enhances Proliferation and Migration in PDGF-BB-Induced Human Aortic Vascular Smooth Muscle Cells

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Abstract

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_0032389wt 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 FRS2wt 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.

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Data Availability

The analyzed datasets generated during the present study are available from the corresponding author upon reasonable request.

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Author notes

  1. Haiyun Qian and Shengwei Ma have contributed to this work as co-first authors.

Authors and Affiliations

  1. Surgical Department of Cardiothoracic Macrovascular, Jingzhou Hospital Affiliated to Yangtze University, No.26 Chuyuan Avenue, Jingzhou District, Jingzhou, 434020, Hubei, China

    Haiyun Qian, Shengwei Ma, Qian Zhou & Chengang Lei

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  1. Haiyun Qian
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  4. Chengang Lei
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Contributions

Conceptualization and Methodology: Shengwei Ma and Qian Zhou; Formal analysis and data curation: Qian Zhou and Chengang Lei; Validation and Investigation: Haiyun Qian and Shengwei Ma; Writing—original draft preparation and Writing—review and editing: Haiyun Qian, Shengwei Ma, and Qian Zhou; Approval of final manuscript: all authors.

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Correspondence to Haiyun Qian.

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Supplementary Information

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12012_2024_9833_MOESM1_ESM.tif

Supplementary Fig. 1 Effects of hsa_circ_0032389, miR-513a-5p and FRS2 on the expression of phenotypic switching markers. (A) SM22α and α-actin protein levels were examined by WB in HA-VSMCs co-transfected with sh-hsa_circ_0032389#1 and anti-miR-513a-5p, and then treated with PDGF-BB (n = 3). (B) SM22α and α-actin protein levels were tested using WB in HA-VSMCs were co-transfected with miR-513a-5p mimic and FRS2 overexpression vector, and then treated with PDGF-BB (n = 3). *P < 0.05.Supplementary file1 (TIF 1181 KB)

12012_2024_9833_MOESM2_ESM.tif

Supplementary Fig. 2 Effects of hsa_circ_0032389/anti-miR-513a-5p and FRS2 on PDGF-BB-induced HA-VSMCs function. (A) FRS2 protein expression was measured by WB analysis in HA-VSMCs co-transfected with hsa_circ_0032389 and sh-FRS2 (n = 3). (B-E) HA-VSMCs were co-transfected with hsa_circ_0032389 and sh-FRS2, and then treated with PDGF-BB (n = 3). Cell proliferation and migration were assessed by CCK8 assay (B), EdU assay (C), transwell assay (D) and wound healing assay (E). (F) FRS2 protein expression was measured by WB analysis in HA-VSMCs co-transfected with anti-miR-513a-5p and sh-FRS2 (n = 3). (G-J) HA-VSMCs were co-transfected with anti-miR-513a-5p and sh-FRS2, and then treated with PDGF-BB (n = 3). CCK8 assay (G), EdU assay (H), transwell assay (I) and wound healing assay (J) were performed to test cell proliferation and migration. *P < 0.05.Supplementary file2 (TIF 1580 KB)

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Qian, H., Ma, S., Zhou, Q. et al. Hsa_circ_0032389 Enhances Proliferation and Migration in PDGF-BB-Induced Human Aortic Vascular Smooth Muscle Cells. Cardiovasc Toxicol 24, 111–121 (2024). https://doi.org/10.1007/s12012-024-09833-w

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