Liposomal UHRF1 siRNA shows lung fibrosis treatment potential through regulation of fibroblast activation

Abstract

Pulmonary fibrosis is a chronic and progressive interstitial lung disease associated with the decay of pulmonary function, which leads to a fatal outcome. As an essential epigenetic regulator of DNA methylation, the involvement of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) in fibroblast activation remains largely undefined in pulmonary fibrosis. In the present study, we found that TGF-β1-mediated upregulation of UHRF1 repressed beclin 1 via methylated induction of its promoter, which finally resulted in fibroblast activation and lung fibrosis both in vitro and in vivo. Moreover, knockdown of UHRF1 significantly arrested fibroblast proliferation and reactivated beclin 1 in lung fibroblasts. Thus, intravenous administration of UHRF1 siRNA-loaded liposomes significantly protected mice against experimental pulmonary fibrosis. Accordingly, our data suggest that UHRF1 might be a novel potential therapeutic target in the pathogenesis of pulmonary fibrosis.

Keywords: Fibrosis; Pulmonology.

Figure 1. UHRF1 is overexpressed in TGF-β1–stimulated fibroblasts and fibrotic lungs.

(A–C) Western blot and corresponding densitometry analysis of fibronectin, collagen I, α-SMA, and UHRF1 in TGF-β1–treated (0, 1, 2, 5 ng/mL for 48 hours) MRC-5 cells and PLFs. Data are shown as the mean ± SEM (n = 3 in each group). (D) Immunofluorescence staining of UHRF1 in MRC-5 cells. Red represents UHRF1; blue represents nuclear DNA staining by DAPI. (E) Immunofluorescence staining of UHRF1 in mouse lung tissues. Red represents UHRF1; blue represents DAPI. (F) Representative results of H&E and UHRF1 IHC staining in lung sections from patients with silicosis and idiopathic pulmonary fibrosis (IPF) and normal participants. (G and H) Western blot and densitometric analysis of fibronectin, collagen I, α-SMA, and UHRF1 protein in saline- or silica-treated mouse lung tissues. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 25 μm (D); 100 μm (E and F). P values were from (B and C) a 1-way ANOVA post hoc test with Tukey’s correction or (H) 2-tailed unpaired Student’s t test.

Figure 2. UHRF1 regulates TGF-β1–induced lung fibroblast proliferation.

(A) qRT-PCR analysis of UHRF1 expression in MRC-5 cells and PLFs transfected with UHRF1 siRNA or its negative control (NC) siRNA, Data are shown as the mean ± SEM (n = 3 in each group). (B and C) Western blot and corresponding densitometry analysis of fibronectin, collagen I, and α-SMA in MRC-5 cells and PLFs transfected with UHRF1 siRNA and its negative control siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). (D) The expression of α-SMA was detected by immunofluorescence staining in MRC-5 cells transfected with UHRF1 siRNA and its negative control siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. (E) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (F and G) Proliferation of MRC-5 cells transfected with UHRF1 siRNA and its negative control siRNA, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (H) Effect of UHRF1 siRNA and its negative control siRNA on the contractility of TGF-β1–induced fibroblasts. Scale bar: 50 μm (D); 100 μm (F). (A, C, E, and G) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 3. The YAP/TEAD pathway contributes to the expression of UHRF1 in fibroblast activation.

(A) The potential binding sites (including TEAD1 and TEAD4) at the UHRF1 promoter region by using the JASPAR database. (B) Target siRNA transfection significantly decreased the expression of TEAD1, TEAD4, and YAP in MRC-5 cells. Data are shown as the mean ± SEM (n = 3 in each group). (C) qRT-PCR detection of UHRF1 expression in MRC-5 cells after transfection with TEAD1, TEAD4, and YAP siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (D–G) Western blot and corresponding densitometry analysis of TEAD1, TEAD4, YAP, and UHRF1 in MRC-5 cells and PLFs transfected with TEAD1, TEAD4, and YAP siRNA and their negative control (NC) siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). (H) Chromatin was harvested for immunoprecipitation with IgG, an anti-TEAD1 antibody, an anti-TEAD4 antibody, an anti-YAP antibody, and an anti–histone H3 antibody. The expression of UHRF1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). P values were from (B) a 2-tailed unpaired Student’s t test and (C and E–H) a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 4. UHRF1 epigenetically mediates beclin 1 methylation in lung fibroblasts.

(A) Prediction of beclin 1 methylation CpG sites by using http://www.urogene.org/methprimer/ (B) Beclin 1 expression in MRC-5 cells and PLFs before and after 5-aza-2′-deoxycytidine treatment. Data are shown as the mean ± SEM (n = 3 in each group). (C) CpG island methylation status of the beclin 1 gene analyzed by a methylation-specific PCR assay in UHRF1 siRNA–treated PLFs. (D) A bisulfite sequencing assay was performed to reveal the CpG methylation status in the beclin 1 promoter region in the PLFs. (E) Chromatin was harvested for immunoprecipitation with IgG, an anti-UHRF1 antibody, after being transfected with TGF-β1 or TGF-β1 plus UHRF1 siRNA. The expression of beclin 1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). (F) Chromatin was harvested for immunoprecipitation with IgG, an anti-Dnmt1 antibody, and an anti–histone H3 antibody after TGF-β1 treatment in the fibroblasts. The expression of beclin 1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). (G) qRT-PCR analysis of beclin 1 expression in MRC-5 cells and PLFs after transfection with UHRF1 siRNA or its negative control (NC) siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (H and I) Western blot and corresponding densitometry analysis of UHRF1 and beclin 1 in MRC-5 cells and PLFs transfected with UHRF1 siRNA and its negative control (NC) siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). P values were from (E) a 2-tailed unpaired Student’s t test and (B, F, G, and I) a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 5. Beclin 1 is a functional downstream gene of UHRF1 and negatively regulates cell proliferation.

(A) qRT-PCR analysis of beclin 1 expression in MRC-5 cells and PLFs after treatment with TGF-β1. Data are shown as the mean ± SEM (n = 3 in each group). (B–D) Western blot and corresponding densitometry analysis of fibronectin, collagen I, α-SMA, and beclin 1 in beclin 1 siRNA–treated MRC-5 cells and PLFs or those treated with its negative control (NC) siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (E) Immunohistochemical staining of α-SMA in MRC-5 cells after beclin 1 siRNA or its negative control siRNA treatment. Red represents α-SMA; blue represents DAPI. (F) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (G) Collagen gel contraction assay was performed to detect the effect of beclin 1 siRNA and its negative control siRNA on the contractility of fibroblasts. (H and I) Proliferation of MRC-5 cells transfected with beclin 1 siRNA and its negative control siRNA, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (J) CCK8 assays were performed to evaluate cell proliferative ability in fibroblasts. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 50 μm (E and H). P values were from (A) a 2-tailed unpaired Student’s t test and (C, D, F, I, and J) a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 6. Loss and gain of function of beclin 1 reversed the effect of UHRF1 in fibroblasts.

(A–C) Western blot and corresponding densitometry analysis of UHRF1, fibronectin, collagen I, α-SMA, and beclin 1 in the MRC-5 cells and PLFs for the indicated groups. Data are shown as the mean ± SEM (n = 3 in each group). (D) Immunohistochemical staining of α-SMA in MRC-5 cells for the indicated groups. α-SMA stained red; DAPI stained blue. (E) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (F and G) Proliferation of MRC-5 cells transfected with different treatment, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (H) CCK8 assays were performed to evaluate ability of MRC-5 cells and PLFs to proliferate. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 50 μm (D and F). (B, C, E, G, and H) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 7. Characterization of UHRF1 siRNA–loaded liposomes.

(A) The hydrodynamic diameter, PDI, ζ potential of the liposomes, and siRNA entrapment efficiency of UHRF1 siRNA–loaded liposomes. (B–D) Distribution of liposome size (B), fluorescence intensity (C), and stability of liposomes (D) loaded in UHRF1 siRNA–loaded liposomes. (E) Representative transmission electron microscopy image of UHRF1 siRNA–loaded liposomes. Scale bar: 200 nm. (F) Representative IVIS images of a mouse at different time points (0 hours, 24 hours and 48 hours) after the administration of DiR-labeled liposomes. (G) Ex vivo fluorescence images of major organs from mice.

Figure 8. Administration of UHRF1 siRNA liposomes attenuates silica-induced pulmonary fibrosis in mice.

(A) Strategy for UHRF1 siRNA–loaded liposome administration in the silica-induced pulmonary fibrosis mouse model. (B) H&E, Sirius red, and Masson’s trichrome staining assays were performed to measure the severity of the lung fibrosis. (C) Semiquantitative Ashcroft scores indicating the severity of fibrosis. Data are shown as the mean ± SEM (n = 6 in each group). (D) The hydroxyproline content in the lungs of mice for the different groups. Data are shown as the mean ± SEM (n = 6 in each group). (E) Western blot of UHRF1, fibronectin, collagen I, α-SMA, and beclin 1 in mouse lung tissues. (F and G) Immunohistochemical staining of collagen I and α-SMA in mouse lung tissues for the indicated groups. Collagen I stained blue; α-SMA stained red; DAPI stained blue. Scale bar: 100 μm (B, F, and G). (C and D) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

Figure 9. Administration of UHRF1 siRNA liposomes attenuates BLM-induced pulmonary fibrosis in mice.

(A) Strategy for UHRF1 siRNA–loaded liposome administration in the BLM-induced pulmonary fibrosis mouse model. (B) H&E, Sirius red, and Masson’s trichrome staining assays were performed to measure the severity of fibrotic lesions. (C) The severity of fibrosis was evaluated by Ashcroft scores. Data are shown as the mean ± SEM (n = 6 in each group). (D) Lungs of mice following different treatments were analyzed for hydroxyproline content. Data are shown as the mean ± SEM (n = 6 in each group). (E) Western blot of UHRF1, fibrotic markers, and beclin 1 in mouse lung tissues in the different groups. (F and G) The expression of collagen I and α-SMA was detected by immunofluorescence staining in mouse lung tissues. Collagen I stained blue; α-SMA stained red; DAPI stained blue. Scale bar: 100 μm (B, F, and G). (C and D) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.