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CircPDE7B/miR-661 axis accelerates the progression of human keloid fibroblasts by upregulating fibroblast growth factor 2 (FGF2)

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Abstract

Circular RNAs (circRNAs) are implicated in keloidogenesis and development. We aimed to investigate the role of a new identified phosphodiesterase 7B-derived circRNA (hsa_circ_0002198; henceforth named as PDE7B) in human keloid fibroblasts (HKFs) and to further confirm its mechanism via competing endogenous RNA (ceRNA) network. Transcriptional and translational levels of circPDE7B, microRNA (miR)-661, fibroblast growth factor 2 (FGF2), cleaved caspase3, B-cell lymphoma (bcl)-2, and bcl-2-associated X protein (bax) were detected by real-time quantitative PCR and western blotting. Relationship among circPDE7B, miR-661, and FGF2 was confirmed by bioinformatics algorithm, dual-luciferase reporter assay, RNA immunoprecipitation, RNA pull-down assay, and Spearman’s rank correlation analysis. Cell progression was measured by cell counting kit-8 assay, 5-ethynyl-2-deoxyuridine assay, transwell assays, and flow cytometry. Expression of circPDE7B was upregulated in human keloid tissues and HKFs, accompanied with miR-661 downregulation and FGF2 upregulation. High circPDE7B accelerated proliferation, migration, and invasion, and inhibited apoptosis. These effects were paralleled with increased bcl-2 and decreased cleaved caspase3 and bax. Moreover, low circPDE7B played opposite effects to high circPDE7B. Restoring miR-661 could suppress HKFs progression, while blocking miR-661 could facilitate that. Notably, miR-661 was directly sponged by circPDE7B and then directly governed FGF2 gene expression. Deleting miR-661 and re-expressing FGF2 both abrogated the suppression of circPDE7B knockdown in HKFs progression. In conclusion, circPDE7B might contribute to HKFs progression via functioning as ceRNA for miR-661, suggesting a novel circPDE7B/miR-661/FGF2 pathway underlying keloid formation and treatment.

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Data availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

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Authors and Affiliations

  1. Department of Plastic Surgery, The First Hospital of Qinhuangdao, No. 258, Wenhua Road, Qinhuangdao, 066000, Hebei, China

    Fenglian Wu, Hongbin He & Jiaxin Wang

  2. Department of Pathology, The First Hospital of Qinhuangdao, Qinhuangdao, China

    Yanxin Chen

  3. Department of Plastic Surgery, The First Hospital of Qinhuangdao, Qinhuangdao, China

    Donglai Zhu

  4. Medical Department, The First Hospital of Qinhuangdao, Qinhuangdao, China

    Tao Jiang

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Contributions

Conceptualization and Methodology: LW and YC; Formal analysis and Data curation: DZ, TJ, and JW; Validation and Investigation: FW, LW, and YC; Writing—original draft preparation and Writing—review and editing: FW, LW, and YC; Approval of final manuscript: all authors.

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Correspondence to Jiaxin Wang.

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The present study was approved by the ethical review committee of The First Hospital of Qinhuangdao.

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Supplementary Information

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11010_2021_4345_MOESM1_ESM.tif

Supplementary Figure 1. There was no direct interaction between circPDE7B and FGF2. RIP assay was performed and RT-qPCR detected relative circPDE7B level in the precipitated complex mediated by anti-FGF2 or anti-IgG in KEL-FIB cells. Supplementary file1 (TIF 200 KB)

11010_2021_4345_MOESM2_ESM.tif

Supplementary Figure 2. Silencing FGF2 retarded HKFs progression. KEL-FIB cells were transfected with si-NC or si-FGF2. (A) Western blotting examined relative FGF2 protein expression. (B) CCK-8 assay examined OD450 values of transfected KEL-FIB cells during 72 h. (C) EdU assay determined positive stained cell percent. (D, E) Transwell assays determined migrated cell numbers and invaded cell numbers after transfection. (F) FCM evaluated apoptosis rate after transfection. (G) Western blotting examined relative protein expression of cleaved-caspase3, bcl-2 and bax in transfected KEL-FIB cells. **P<0.01 and ***P<0.001. Supplementary file2 (TIF 6255 KB)

Supplementary file. The images of EdU assay. Supplementary file3 (PDF 538 KB)

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Wu, F., He, H., Chen, Y. et al. CircPDE7B/miR-661 axis accelerates the progression of human keloid fibroblasts by upregulating fibroblast growth factor 2 (FGF2). Mol Cell Biochem 477, 1113–1126 (2022). https://doi.org/10.1007/s11010-021-04345-5

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