LncRNA CTD-2528L19.6 prevents the progression of IPF by alleviating fibroblast activation

Abstract

Long non-coding RNAs (lncRNAs) have emerged as critical factors for regulating multiple biological processes during organ fibrosis. However, the mechanism of lncRNAs in idiopathic pulmonary fibrosis (IPF) remains incompletely understood. In the present study, two sets of lncRNAs were defined: IPF pathogenic lncRNAs and IPF progression lncRNAs. IPF pathogenic and progression lncRNAs-mRNAs co-expression networks were constructed to identify essential lncRNAs. Network analysis revealed a key lncRNA CTD-2528L19.6, which was up-regulated in early-stage IPF compared to normal lung tissue, and subsequently down-regulated during advanced-stage IPF. CTD-2528L19.6 was indicated to regulate fibroblast activation in IPF progression by mediating the expression of fibrosis related genes LRRC8C, DDIT4, THBS1, S100A8 and TLR7 et al. Further studies showed that silencing of CTD-2528L19.6 increases the expression of Fn1 and Collagen I both at mRNA and protein levels, promoted the transition of fibroblasts into myofibroblasts and accelerated the migration and proliferation of MRC-5 cells. In contrast, CTD-2528L19.6 overexpression alleviated fibroblast activation in MRC-5 cells induced by TGF-β1. LncRNA CTD-2528L19.6 inhibited fibroblast activation through regulating the expression of LRRC8C in vitro assays. Our results suggest that CTD-2528L19.6 may prevent the progression of IPF from early-stage and alleviate fibroblast activation during the advanced-stage of IPF. Thus, exploring the regulatory effect of lncRNA CTD-2528L19.6 may provide new sights for the prevention and treatment of IPF.

Fig. 1. Identification of IPF lncRNAs signatures.

A Venn diagram of overlapping DE lncRNAs among three groups. B, C Distribution of expression levels for key lncRNAs CTD-2528L19.6 and NR2F1-AS1. *P < 0.05, **P < 0.01 in Student’s t-test. D, E Heatmaps show the global expression levels of IPF pathogenic lncRNAs (D) and IPF progression lncRNAs (E) in GSE24206.

Fig. 2. LncRNA-mRNA co-expression networks during the pathogenesis of IPF.

A IPF pathogenic lncRNAs (diamond)-mRNAs (oval) co-expression network in IPF. The nodes marked with red color represent the up-regulated IPF pathogenic genes. The nodes marked with blue color represent the down-regulated IPF pathogenic genes. The solid lines (dotted lines) represent the positive (negative) correlation between mRNAs and lncRNAs (|r| >0.8, P < 0.01, Pearson Correlation Test). B Distribution of the degree of genes in the IPF pathogenic network. C Circos plots display chromosomal interactions between co-expressed lncRNAs and mRNAs. The location of lncRNA CTD-2528L19.6 was marked with red arrows. D, E KEGG pathways enrichment with IPF pathogenic up-regulated mRNAs (D) or down-regulated mRNAs (E). FDR < 0.05, Hypergeometric test. Size of the bubble indicates the number of genes in the corresponding annotation. Color shade corresponds to the -log10(p) value. The closer the color is to red, the more significant the enrichment is.

Fig. 3. IPF co-expression networks in early-stage and advanced-stage of IPF.

A IPF progression lncRNAs (diamond)-mRNAs (oval) co-expression network in early-stage IPF. The solid lines (dotted lines) represent the positive (negative) correlation between mRNAs and lncRNAs (|r| >0.8, P < 0.01, Pearson Correlation Test). The nodes marked with green color represent the early-stage IPF specific genes. The nodes marked with yellow color represent the common genes in early-stage and advanced-stage IPF progression network. Several important IPF biomarkers and fibroblast related mRNAs that co-expressed with CTD-2528L19.6 were marked with red arrows. B, C Distribution of the degree of genes in early-stage IPF progression network. D IPF progression lncRNAs (diamond)-mRNAs (oval) co-expression network in advanced-stage IPF. The nodes marked with blue color represent specific genes in advanced-stage IPF. The nodes marked with yellow color represent the common genes in early-stage and advanced-stage IPF progression network. Several important IPF biomarkers and fibroblast related mRNAs that co-expressed with CTD-2528L19.6 were marked with red arrows. E, F Distribution of the degree of genes in the advanced-stage IPF progression network.

Fig. 4. Illustration of fibrosis related gene sets that contain the mRNAs co-expressed with lncRNAs in the progression of IPF.

A, B Sankey diagrams display the connection between CTD-2528L19.6 correlated mRNAs and fibrosis related gene sets in early-stage IPF (A) and advanced-stage IPF (B). The expression of mRNAs in diagrams are correlated with the expression of CTD-2528L19.6 (|r| >0.8, P < 0.01, Pearson Correlation Test). Path connecting an mRNA to a fibrosis related gene set represents the mRNA participates in the gene set.

Fig. 5. Correlation between CTD-2528L19.6 and six IPF cell markers in IPF patients.

A-F The correlation relationship between CTD-2528L19.6 and six IPF cell markers (KRT5, NGFR, MUC5AC, MUC5B, SCGB1A1, SPDEF) was tested by Pearson correlation test. The fitting model was constructed using the “lm” method function in R package ggplot2.

Fig. 6. Silencing CTD-2528L19.6 promotes fibroblast activation of MRC-5 cells.

A Fluorescence in situ hybridization detects the localization of CTD-2528L19.6 in MRC-5 cells. For clarity, CTD-2528L19.6 was abbreviated as CTD in the figures. 18 S and U6 are the markers of cytoplasm and nuclei, respectively. Blue nuclei are stained with DAPI. Scale bar: 50 μm. B, C The expression of CTD-2528L19.6, Fn1, Collagen 1α1 after silencing CTD-2528L19.6 in MRC-5 cells were detected by qRT-PCR. n = 4. D Western blot was used to detect the expression of Fn1 and Collagen I after SSi-CTD transfection. n = 6. Wound-healing migration assay (E) and EdU proliferation assay (F) were used to assay the effect of SSi-CTD on migration and proliferation in MRC-5 cells. n = 4. Scale bars, 200 μm in E and 50 μm in F. G Immunofluorescence was applied to examine the fibroblasts into myofibroblasts transition after transfecting SSi-CTD. Scale bar, 20 μm. *P < 0.05; **P < 0.01.

Fig. 7. LncRNA CTD-2528L19.6 inhibits the TGF-β1-induced fibroblast activation in MRC-5 cells.

qRT-PCR analysis of the expression of CTD-2528L19.6 (A) and Fn1, Collagen 1α1 (B) in MRC-5 cells after transfection CTD-2528L19.6 under TGF-β1 induced fibroblast activation. n = 4. C Western blot results demonstrate that the overexpression of CTD-2528L19.6 inhibits the pro-fibrotic effect of TGF-β1 in MRC-5. n = 9. Wound-healing assay (D) and EdU proliferation assay (E) reveals the inhibitory effects of CTD-2528L19.6 on TGF-β1-induced migration and proliferation in MRC-5 cells. n = 4. Scale bars, 200 μm in D and 50 μm in E. F Fibroblast to myofibroblast transition was observed by immunofluorescence. Scale bar, 20 μm. *P < 0.05; **P < 0.01.

Fig. 8. LncRNA CTD-2528L19.6 regulates LRRC8C in MRC-5 cells.

A, B qRT-PCR shows that silencing or forced overexpression of CTD-2528L19.6 inhibited (A) or promoted (B) the expression of LRRC8C. C-E Silencing of LRRC8C restored the expression levels of Fn1 and Collagen 1α1 in TGF-β1-treated MRC-5 cells. n = 4; *P < 0.05, **P < 0.01.