CircHIPK3 regulates pulmonary fibrosis by facilitating glycolysis in miR-30a-3p/FOXK2-dependent manner

Abstract

Pulmonary fibrosis develops when myofibroblasts and extracellular matrix excessively accumulate in the injured lung, but what drives fibrosis is not fully understood. Glycolysis has been linked to cell growth and proliferation, and several studies have shown enhanced glycolysis promotes pulmonary fibrosis. However, detailed studies describing this switch remain limited. Here, we identified that TGF-β1 effectively increased the expression of circHIPK3 in lung fibroblasts, and circHIPK3 inhibition attenuated the activation, proliferation, and glycolysis of fibroblasts in vitro. Dual-luciferase reporter gene assays, RNA immunoprecipitation (RIP), and RNA pull-down assays showed that circHIPK3 could function as a sponge of miR-30a-3p and inhibit its expression. Furthermore, FOXK2, a driver transcription factor of glycolysis, was identified to be a direct target of miR-30a-3p. Mechanistically, circHIPK3 could enhance the expression of FOXK2 via sponging miR-30a-3p, thereby facilitating fibroblast glycolysis and activation. Besides, miR-30a-3p overexpression or FOXK2 knockdown blocked fibroblast activation induced by TGF-β1 and abrogated the profibrotic effects of circHIPK3. Moreover, circHIPK3 and miR-30a-3p were also dysregulated in fibrotic murine lung tissues induced by silica. Adeno-associated virus (AAV)-mediated circHIPK3 silence or miR-30a-3p overexpression alleviated silica-induced pulmonary fibrosis in vivo. In conclusion, our results identified circHIPK3/miR-30a-3p/FOXK2 regulatory pathway as an important glycolysis cascade in pulmonary fibrosis.

Keywords: FOXK2; ceRNA; circHIPK3; glycolysis; silicosis.

Figure 1

Glycolysis plays a key role in TGF-β1-induced pulmonary fibroblast activation. (A) Western blot and densitometric analysis of Fibronectin, Collagen I, and α-SMA in MRC-5 cells were treated with 0, 1, 2, 5 ng/ml TGF-β1 for 48h. (B) Immunofluorescence staining of α-SMA in MRC-5 cells for the control and TGF-β1 (5 ng/ml) treatment groups. Red represents α-SMA staining; blue represents nuclear DNA staining by DAPI. (C) DNA synthesis was assessed using EDU assay in MRC-5 cells for the control and TGF-β1 (5 ng/ml) treatment groups. Red, EDU; blue, nuclei. (D) Western blot detected levels of HK2, PFKM, PKM2, and PDK1 in MRC-5 cells were treated with 5 ng/ml TGF-β1 for 0h, 3h, 6h, 12h, 24h, 48h. (E-F) MCR-5 cells were treated with 0, 1, 2, 5 ng/ml TGF-β1 for 48 h. Lactate levels and glucose consumption were determined (n = 3), with *P < 0.05, **P < 0.01 vs. the control group. (G-H) MRC-5 cells were pretreated with 2-DG (1 mM, 3 mM, 10 mM) for 1 hour, followed by TGF-β1 treatment for 48 hours. Fibronectin, Collagen I, and α-SMA expression were determined by western blot, and cell viability was detected by MTT assays (n = 3), **P < 0.01.

Figure 2

CircHIPK3 is involved in TGF-β1-derived fibroblast activation and proliferation. (A) CircHIPK3 and HIPK3 mRNA expression were determined by qRT-PCR in MRC-5 cells treated with 0, 1, 2, 5 ng/ml TGF-β1 for 48h (n = 3), with **P < 0.01 vs. the control group. (B) RT-PCR validated the existence of circHIPK3 in MRC-5 cell lines. CircHIPK3 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. (C-D) The expression of circHIPK3 and HIPK3 mRNA in MRC-5 was detected by RT-PCR or qRT-PCR in the presence or absence of RNase R. (E) The abundance of circHIPK3 and HIPK3 mRNA was assessed by qRT-PCR MRC-5 cells treated with Actinomycin D (2μM) at the indicated time points. (F) Expression of circHIPK3 and HIPK3 in nuclear and cytoplasm of MRC-5 were measured via qRT-PCR analysis. (G) Fluorescence in situ hybridization (FISH) assay was conducted to determine the subcellular localization and expression of circHIPK3 in control and TGF-β1-treated groups. (H) Western blot analysis of Fibronectin, Collagen I, and α-SMA in MRC-5 cells transfected with circHIPK3 siRNA or its negative control then treated with 5ng/ml TGF-β1 for 48h. (I) Immunofluorescence staining of α-SMA in MRC-5 cells for the indicated groups. Red represents α-SMA staining; blue represents nuclear DNA staining by DAPI. (J) EDU assays of MRC-5 cells transfected with control or circHIPK3 siRNA were performed to evaluate cell proliferative ability.

Figure 3

CircHIPK3 acts as a sponge for miR-30a-3p in lung fibroblasts. (A) Schematic illustration exhibiting overlapping of the target miRNAs of circHIPK3 predicted by miRanda, miRDB, circBank, and RNAhybrid. (B) The relative levels of miRNAs in MRC-5 cells were pulled down by a circHIPK3 probe. (C) The relative levels of circHIPK3 in MRC-5 cells were pulled down by biotinylated miR-30a-3p, miR-30d-3p, miR-338-3p and miR-653-5p (n = 3), with **P < 0.01 vs. the Bio-miR-NC group. (D) The miR-30a-3p was down-regulated in IPF patients based on the microarray dataset GSE32538 and GSE27430. (E) MiR-30a-3p expression were determined by qRT-PCR in MRC-5 cells treated with 0, 1, 2, 5 ng/ml TGF-β1 for 48h (n = 3), with **P < 0.01 vs. control group. (F) Schematic of putative binding sites of miR-30a-3p on circHIPK3 transcript. (G) MRC-5 were co-transfected with LUC-circHIPK3-wt, LUC-circHIPK3-mut-1, LUC-circHIPK3-mut-2, or LUC-circHIPK3-mut-1/2 with miR-30a-3p mimics or scrambled mimics. Luciferase activity was detected 24 h after transfection (n = 3), **P < 0.01. (H) RIP assays for circHIPK3 and miR-30a-3p levels in MRC-5 cells were detected by qRT-PCR analysis (n = 3), **P < 0.01. (I) Colocalization between miR-30a-3p and circHIPK3 was observed via FISH assays in MRC-5 cells. CircHIPK3 probes were labeled with Alexa Fluor 555. MiR-30a-3p probes were labeled with Alexa Fluor 488. Nuclei were stained with DAPI.

Figure 4

MiR-30a-3p mediates the function of circHIPK3 to regulate fibroblast activation. (A) Western blot and densitometric analysis of Fibronectin, Collagen I, and α-SMA in MRC-5 cells transfected with miR-30a-3p or control mimic then treated with 5ng/ml TGF-β1 for 48h (n = 3), *P < 0.05. (B) Immunofluorescence staining of Collagen I in MRC-5 cells for the indicated groups. Green represents Collagen I staining; blue represents nuclear DNA staining by DAPI. (C-D) EDU and MTT assays were performed to evaluate cell proliferative ability in MRC-5 cells (n = 3), *P < 0.05, **P < 0.01. (E-G) Western blot analysis of the protein expression of Fibronectin, Collagen I, and α-SMA in treated MRC-5 cells for the indicated groups.

Figure 5

FOXK2 is a functional target of miR-30a-3p and exerts profibrotic effects by regulating glycolysis. (A) Schematic diagram of conserved target sites of miR-30a-3p in the 3'UTR of several species FOXK2 mRNA. (B) Luciferase activities of Luc-FOXK2-wild or Luc-FOXK2-mutant in MRC-5 cells co-transfected with miR-30a-3p mimic, miR-30a-3p inhibitor, or N.C. were determined by a luciferase reporter assay (n = 3), **P < 0.01. (C) Western blot detected levels of FOXK2, Fibronectin, Collagen I, and α-SMA in MRC-5 cells transfected with FOXK2 siRNA or its negative control then treated with 5 ng/ml TGF-β1 for 48 h. (D) Cell proliferation was detected using EDU assay. Red, EDU; blue, nuclei. Scale bar, 100μm. (E) FOXK2, Fibronectin, Collagen I, and α-SMA expression levels were detected by western blot analysis. (F-I) Lactate concentration, glucose consumption, glycolytic rate, and ATP concentration were detected in MRC-5 cells for the indicated groups (n = 3), **P < 0.01.

Figure 6

CircHIPK3 and miR-30a-3p are dysregulated during pulmonary fibrogenesis. (A) The C57BL/6 mice were sacrificed on days 7, 14, and 28 after intratracheal instillation of silica suspended saline and saline. Histological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. (B) Masson staining and IHC staining assays were performed to measure fibrotic lesions and Col-1 expression. (C) Hydroxyproline content of the lung tissues was used to assess the degree of collagen deposition. (D) Western blot and densitometric analysis of the protein expression of Fibronectin, Collagen I, and α-SMA in mouse lung tissues. GAPDH was used as an internal loading control. (E-F) qRT-PCR analysis of circHIPK3 and miR-30a-3p expression in mouse fibrotic lung tissue on days 7, 14, and 28. (G) Lactate concentration was detected in lung tissues exposed to silica for 7, 14, and 28 days (n = 6), with **P < 0.01 vs. the control group.

Figure 7

CircHIPK3 and miR-30a-3p regulate silica-induced pulmonary fibrosis in vivo. (A) Strategy for circHIPK3 knockdown or miR-30a-3p overexpression or in bleomycin silica-induced pulmonary fibrosis mouse model. (B) MiR-30a-3p and circHIPK3 expression were measured by qRT-PCR in the treated mouse lung tissues for the indicated groups. (C) H&E staining, Masson staining, and IHC staining of Collagen I were performed to measure the severity of the lung fibrosis. (D) MiR-30a-3p overexpression or circHIPK3 silence attenuated the hydroxyproline content in the lungs of mice treated with SiO₂. (E) The protein expression of Fibronectin, Collagen I, and α-SMA in mouse lung tissues treated was determined by western blot. GAPDH was used as an internal loading control. (F) circHIPK3 silence or miR-30a-3p overexpression decreased the lactate levels in the lungs of mice treated with SiO₂. (G) Foxk2, HK2, PFKM, PKM2, and PDK1 were detected by western blot analysis. GAPDH was used as an internal loading control.