Angelica Sinensis Polysaccharide Suppresses Epithelial-Mesenchymal Transition and Pulmonary Fibrosis via a DANCR/AUF-1/FOXO3 Regulatory Axis

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of lung fibroblasts and extracellular matrix deposition. Angelica sinensis polysaccharide (ASP), the major bioactive component that can extracted from roots of angelica, plays functional roles in immunomodulation, anti-tumor activity, and hematopoiesis. Emerging evidence has suggested that long noncoding RNAs (lncRNAs) play important roles in pathophysiological processes in various diseases. However, the roles of lncRNAs and ASP in IPF remain poorly understood. In the present study, we investigated the effects of ASP in IPF, as well as their functional interactions with lncRNA DANCR (differentiation antagonizing non-protein coding RNA). IPF models were established by treating Sprague-Dawley rats with BLM and treating alveolar type Ⅱ epithelial (RLE-6TN) cells with TGF-β1. Our results showed that ASP treatment suppressed pulmonary fibrosis in rats and fibrogenesis in RLE-6TN cells. The lncRNA DANCR is downregulated after ASP treatment in both rat lung tissues and RLE-6TN cells, and DANCR overexpression dramatically reversed the suppressive effects of ASP in IPF. Mechanistically, DANCR directly binds with AUF1 (AU-binding factor 1), thereby upregulating FOXO3 mRNA and protein levels. Moreover, overexpression of AUF1 or FOXO3 reversed the functional effects induced by ASP treatment. In conclusion, our findings showed that DANCR mediates ASP-induced suppression of IPF via upregulation of FOXO3 protein levels in an AUF1-dependent manner. Therefore, DANCR could serve as a promising therapeutic target in IPF treatment with ASP.

Keywords: AUF1; DANCR; FOXO3; angelica sinensis polysaccharide; idiopathic pulmonary fibrosis.

Figure 1.

ASP treatment inhibits pulmonary fibrosis in vivo and cell fibrogenesis in vitro. (A) H&E and Masson’s staining assays were performed to demonstrate the effects of BLM and ASP on pulmonary fibrosis of rat lung tissues. Immunohistochemical analysis of collagen-1 protein levels in rat lung tissues. (B) Morphological changes in RLE-6TN cells, such as disappearance of intercellular junction and spindle-like structures (indicated by arrows), were observed after treatment with TGF-β1. (C) Cell proliferation was evaluated by performing CCK8 assay in the different sample groups, ^*P<0.05. (D) Transwell assay was performed to detect the migration of RLE-6TN cells, ^*P<0.05. (E) The expression levels of E-cadherin and α-SMA in RLE-6TN cells were measured by western blotting. Protein expression levels relative to β-actin levels are shown, ^**P<0.01.

Figure 2.

LncRNA DANCR is essential for ASP-induced suppression of fibrogenesis. (A-B) qRT-PCR was performed to determine the expression levels of DANCR in RLE-6TN cells (A) and rat lung tissues (B), which are treated with or without ASP, ^*P<0.05, ^**P<0.01. (C) DANCR was overexpressed in RLE-6TN cells by infection with DANCR-specific plasmid, ^***P<0.001. (D) CCK8 assay revealed that DANCR overexpression attenuated ASP-induced suppression of cell proliferation, ^*P<0.05. (E) Results of Transwell assay showed that ASP suppressed cell migration of RLE-6TN cells; however, transfection with DANCR reversed this effect, ^*P<0.05. (F) Immunofluorescence assays showed that upregulation of DANCR levels abrogated the ASP-induced inhibition of collagen-1, E-cadherin, and α-SMA expression. Green fluorescence represents respective proteins (collagen-1, E-cadherin, and α-SMA), and blue fluorescence indicates nuclei stained by DAPI. (G) qPCR analysis was performed to evaluate the upregulation of DANCR levels in rats administered with p-DANCR compared to those in control rats, ^**P<0.01. (H) H&E and Masson’s staining assays were performed to investigate the effects of DANCR in ASP-induced suppression of pulmonary fibrosis in rat lung tissues; immunohistochemical analysis of collagen-1 protein expression in rat lung tissues overexpressing DANCR.

Figure 3.

DANCR regulates fibrogenesis by inducing FOXO3 expression. (A) Heatmap showing mRNA expression levels in RLE-6TN cells transfected with control or DANCR plasmid for 48 h. Arrow indicates FOXO3. (B) FOXO3 expression was measured by western blot analysis in cells overexpressing DANCR. (C) FOXO3 protein levels in rat lung tissues were analyzed by immunohistochemistry. (D) FOXO3 mRNA expression was detected in RLE-6TN cells (left panel) and rat lung tissues (right panel) by qRT-PCR. (E) Immunohistochemical analysis of FOXO3 levels in lung tissues treated with ASP or PBS control. (F) Western blot assay was performed to detect FOXO3 protein levels in RLE-6TN cells treated with ASP or PBS control. (G) FOXO3 expression was silenced in RLE-6TN cells by transfection with FOXO3 siRNA, ^**P<0.01. (H) Relative cell proliferation was measured by CCK8 assay in cells overexpressing DANCR and (or) FOXO3 knockdown cells, ^*P<0.05. (I) Cell migration was evaluated by Transwell assay in cells overexpressing DANCR and (or) FOXO3 knockdown cells, ^**P<0.01. (J) Effect of FOXO3 and DANCR on the expression of EMT-related proteins were identified by western blotting.

Figure 4.

LncRNA DANCR is associated with AUF1 in RLE-6TN cells. (A) Nuclear fraction experiments and qRT-PCR experiments were performed to determine the relative distribution of DANCR in nucleus and cytoplasm of RLE-6TN cells. (B) The distribution of DANCR was determined by performing RNA fluorescence in situ hybridization (FISH) in RLE-6TN cells. (C) Prediction of 350-670-nt DANCR structures based on minimum free energy (MFE) and partition function (http://rna.tbi.univie.ac.at/). (D) Immunofluorescence assay was performed to identify the subcellular distribution of AUF1 proteins in RLE-6TN cells. (E) RNA pulldown followed by western blotting was performed with DANCR probe to verify the direct interaction between AUF1 and DANCR. GAPDH was used as the internal control. (F) RNA immunoprecipitation (RIP) was performed with an anti-AUF1 antibody to identify the association between DANCR and AUF1. Enrichment was shown as the percentage of input, ^***P<0.001.

Figure 5.

DANCR promotes translation of FOXO3 gene by binding to AUF1. (A) RIP was performed using anti-AUF1 and control IgG antibodies, followed by qRT-PCR to determine the enrichment of DANCR and U6. U6 was used as the negative control, ^**P<0.01. (B) Experimental validation of AUF1 knockdown in RLE-6TN cells by qRT-PCR and western blotting, ^***P<0.001. (C) mRNA and protein levels of FOXO3 in RLE-6TN cells silenced with AUF1. (D) Western blotting was performed to detect the protein expression levels of FOXO3 in RLE-6TN cells overexpressing DANCR or (and) silenced with AUF1. (E) RLE-6TN lysates were incubated with in vitro-synthesized, biotin-labeled control LacZ DNA probes or DNA probes against DANCR for the biotinylated oligonucleotide pulldown assay. ^**P<0.01 compared to respective LacZ probes. (F) Endogenous AUF1 binding to FOXO3 mRNA was modified by DANCR overexpression, ^**P<0.01. (G) Control plasmids or DANCR plasmids transfected RLE-6TN cells were cultured with 20 μg/ml cycloheximide (CHX) for 0-150 min and analyzed by western blotting.

Figure 6.

ASP suppresses fibrogenesis by regulating the DANCR/AUF1/FOXO3 axis. (A) AUF1 overexpression was validated by qRT-PCR and western blotting, ^***P<0.001. (B) Cell proliferation was measured by CCK8 assay in cells treated with ASP and (or) p-AUF1, ^*P<0.05. (C) Cell migration was evaluated by Transwell assay in cells treated with ASP and(or) p-AUF1, ^*P<0.05. (D) Expression of EMT-relevant proteins, E-cadherin, and α-SMA were detected by western blot experiments in cells treated with ASP and (or) AUF1 plasmid. (E) Cell proliferation was measured by CCK8 assay in cells treated with ASP and (or) p-AUF1, ^*P<0.05. (F) Cell migration was evaluated by Transwell assay in cells treated with ASP and (or) p-AUF1, ^*P<0.05. (G) Expression levels of EMT-relevant proteins, E-cadherin, and α-SMA, were detected by western blot experiment in cells treated with ASP and (or) AUF1 plasmid.

Figure 7.

Schematic showing the proposed regulatory mechanisms of ASP. ASP treatment upregulated DANCR expression. DANCR overexpression upregulates FOXO3 expression by guiding AUF1 to activate the translation of FOXO3 mRNA, thereby promoting proliferation, migration, and the EMT and initiation of pulmonary fibrosis.