Circular RNA hsa_circ_0043688 serves as a competing endogenous RNA for microRNA-145-5p to promote the progression of Keloids via Fibroblast growth factor-2

Abstract

Background: Keloids are benign fibroproliferative skin tumors. Circular RNA (circRNA) hsa_circ_0043688 has been exhibited to the freakishly expressed in keloid tissues. Here, we aimed to investigate the regulatory network of hsa_circ_0043688 in the pathological process of keloid.

Methods: Hsa_circ_0043688, microRNA-145-5p (miR-145-5p), and Fibroblast growth factor-2 (FGF2) level were detected using RT-qPCR. Cell viability, proliferation, apoptosis, invasion, and migration were investigated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays, respectively. Western blot analysis of protein levels of FGF2, CyclinD1, Collagen I, and Collagen III. After the prediction of Circinteractome and Starbase, their interaction was verified based on a dual-luciferase reporter and RIP assays.

Results: Increased hsa_circ_0043688 and FGF2, and decreased miR-145-5p in keloids samples and fibroblasts were found. Also, hsa_circ_0043688 absence hindered proliferation, invasion, migration, and boost apoptosis of keloid fibroblasts. In mechanism, hsa_circ_0043688 modulated FGF2 content via sponging miR-145-5p.

Conclusion: Hsa_circ_0043688 knockdown inhibited cell growth and metastasis of keloid fibroblasts via miR-145-5p/FGF2, providing a new mechanism to understand the keloid progression.

Keywords: FGF2; Hsa_circ_0043688; keloid fibroblasts; miR-145-5p; proliferation.

FIGURE 1

Hsa_circ_0043688 expression in keloid. (A and B) RT‐qPCR analysis of hsa_circ_0043688 content in 51 normal skin tissues, 51 keloid tissues, normal fibroblasts, and keloid fibroblasts. (C) Subcellular fractionation analysis of hsa_circ_0043688 cellular localization in keloid fibroblasts. (D) Effects of RNase R treatment on hsa_circ_0043688 and linear FKBP10 in keloid fibroblasts. ***p < 0.001, ****p < 0.0001

FIGURE 2

Effects of hsa_circ_0043688 downregulation on the malignant behavior of keloid fibroblasts. Keloid fibroblasts were transfected with si‐NC or si‐hsa_circ_0043688. (A) RT‐qPCR analysis of hsa_circ_0043688 content in transfected keloid fibroblasts. (B and C) Cell proliferation was analyzed in transfected keloid fibroblasts by CCK‐8 and EdU assay. (D) Apoptosis rates were examined in transfected keloid fibroblasts by flow cytometry assay. (E and F) Transwell and wound healing analysis of invasion and migration in transfected keloid fibroblasts. (G) Protein levels of CyclinD1, Collagen I, and Collagen III were determined in transfected keloid fibroblasts using western blot assay. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 3

Hsa_circ_0043688 sequestered miR‐145‐5p in keloid fibroblasts. (A) The mutant and putative binding site of hsa_circ_0043688 with miR‐145‐5p. (B) miR‐145‐5p level was determined in miR‐NC or miR‐145‐5p‐transfected keloid fibroblasts by RT‐qPCR assay. (C and D) Their binding was verified using a dual‐luciferase reporter and RIP assay. (E) RT‐qPCR analysis of miR‐145‐5p content keloid tissues. (F) The expression association between hsa_circ_0043688 and miR‐145‐5p in keloid tissues was analyzed by Pearson correlation analysis. (G) RT‐qPCR analysis of miR‐145‐5p in keloid fibroblasts. (H) Hsa_circ_0043688 level was determined in pCD5‐ciR or hsa_circ_0043688‐transfected keloid fibroblasts by RT‐qPCR assay. (I) Effects of hsa_circ_0043688 upregulation or silencing on miR‐145‐5p content were examined in keloid fibroblasts using RT‐qPCR assay. **p < 0.01, ****p < 0.0001

FIGURE 4

Effects of miR‐145‐5p on hsa_circ_0043688‐regulated proliferation, apoptosis, invasion, and migration of keloid fibroblasts. Keloid fibroblasts were transfected with si‐NC, si‐hsa_circ_0043688, si‐hsa_circ_0043688 + anti‐miR‐NC, and si‐hsa_circ_0043688 + anti‐miR‐145‐5p. (A) RT‐qPCR analysis of miR‐145‐5p content in transfected keloid fibroblasts. (B and C) CCK‐8 assay and EdU analysis of keloid fibroblasts proliferation ability. (D and E) Apoptosis rate in transfected keloid fibroblasts was assessed. (F and G) Transwell and wound healing assays were carried out to examine the capacities of invasion and migration in transfected keloid fibroblasts. (H) Western blot assay was conducted to test CyclinD1, Collagen I, and Collagen III protein levels in transfected keloid fibroblasts. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 5

FGF2 acted as a target of miR‐145‐5p in keloid fibroblasts. (A) The sequences of FGF2 mRNA 3’UTR including wild type and mutant are shown with the miR‐145‐5p sequence. (B) A dual‐luciferase reporter assay was used to examine the relative luciferase activity in keloid fibroblasts. (C) The endogenous association was analyzed by RIP assay. (D) FGF2 level was determined in normal skin tissues and keloid tissues by RT‐qPCR assay. (E) Pearson correlation analysis was applied to evaluate the expression association between FGF2 and miR‐145‐5p in keloid tissues. (F) FGF2 protein level was examined in normal skin tissues and keloid tissues by western blot assay. (G) Western blot analysis of FGF2 protein level in normal fibroblasts and keloid fibroblasts. (H) Knockdown efficiency of anti‐miR‐145‐5p in keloid fibroblasts was assessed. (I) FGF2 content was determined in keloid fibroblasts transfected with miR‐NC, miR‐145‐5p, anti‐miR‐NC, and anti‐miR‐145‐5p by western blot assay. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 6

miR‐145‐5p inhibited proliferation, invasion, migration, and induced apoptosis of keloid fibroblasts by interacting with FGF2. Keloid fibroblasts were transfected with miR‐NC, miR‐145‐5p, miR‐145‐5p + pcDNA, and miR‐145‐5p + FGF2. (A) Western blot analysis of FGF2 in transfected keloid fibroblasts. (B and C) The proliferation ability of keloid fibroblasts was analyzed by CCK‐8 assay and EdU assay. (D and E) Apoptosis rate of keloid fibroblasts was assessed by flow cytometry assay. (F and G) The capacities of invasion and migration in keloid fibroblasts were examined by transwell and wound healing assay. (H) CyclinD1, Collagen I, and Collagen III protein levels of keloid fibroblasts were determined by RT‐qPCR assay and western blot assay. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 7

FGF2 was positively regulated by hsa_circ_0043688/miR‐145‐5p. (A and B) FGF2 level was detected in keloid fibroblasts transfected with si‐NC, si‐hsa_circ_0043688, si‐hsa_circ_0043688 + anti‐miR‐NC, and si‐hsa_circ_0043688 + anti‐miR‐145‐5p by western blot assay. **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 8

Hsa_circ_0043688 could regulate proliferation, migration, invasion, and ECM production of keloid fibroblasts by targeting the miR‐145‐5p/FGF2 axis