Genome-wide CRISPR/Cas9 screening identifies key profibrotic regulators of TGF-β1-induced epithelial-mesenchymal transformation and pulmonary fibrosis

Abstract

Background: The idiopathic pulmonary fibrosis (IPF) is a progressive and lethal interstitial lung disease with high morbidity and mortality. IPF is characterized by excessive extracellular matrix accumulation (ECM) and epithelial-mesenchymal transformation (EMT). To date, few anti-fibrotic therapeutics are available to reverse the progression of pulmonary fibrosis, and it is important to explore new profibrotic molecular regulators mediating EMT and pulmonary fibrosis.

Methods: Based on our model of TGF-β1-induced EMT in BEAS-2B cells, we performed the genome-wide CRISPR/Cas9 knockout (GeCKO) screening technique, pathway and functional enrichment analysis, loss-of-function experiment, as well as other experimental techniques to comprehensively investigate profibrotic regulators contributing to EMT and the pathogenesis of pulmonary fibrosis.

Results: Utilizing the GeCKO library screening, we identified 76 top molecular regulators. Ten candidate genes were subsequently confirmed by integrating the high-throughput data with findings from pathway and functional enrichment analysis. Among the candidate genes, knockout of COL20A1 and COL27A1 led to decreased mRNA expression of ECM components (Fibronectin and Collagen-I), as well as an increased rate of cell apoptosis. The mRNA expression of Collagen-I, together with the cell viability and migration, were inhibited when knocking out the WNT11. In addition, a decrease in the protein deposition of ECM components was observed by suppressing the expression of COL20A1, COL27A1, and WNT11.

Conclusion: Our study demonstrates that the COL20A1, COL27A1, and WNT11 serve as key profibrotic regulators of EMT. Gaining understanding and insights into these key profibrotic regulators of EMT paves the way for the discovery of new therapeutic targets against the onset and progression of IPF.

Keywords: COL20A1; COL27A1; TGF-β1; WNT11; epithelial-mesenchymal transformation; extracellular matrix; genome-wide CRISPR/Cas9 knockout screening; idiopathic pulmonary fibrosis.

FIGURE 1

TGF-β1 treatment induces the EMT and ECM-generating phenotype in BEAS-2B and BEAS-2B-GeCKO cells. (A) The mRNA expression of Collagen-I and Fibronectin in BEAS-2B cells was determined by RT-qPCR following treatment with different doses of rTGF-β1. The optimized concentration of rTGF-β1 (10 ng/mL) was confirmed for subsequent experiments. (B) The mRNA expression of Collagen-I and Fibronectin in BEAS-2B-GeCKO cells was determined by RT-qPCR following rTGF-β1 treatment. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA with Tukey’s multiple comparisons test (A) or unpaired two-tailed t-test (B). In (A), n = 6 for the control group; n = 3 for rTGF-β1 treated groups; In (B), n = 3 per group. * indicates significant difference compared with the control. *P < 0.05, **P < 0.01, ***P < 0.001. GeCKO, Genome-wide CRISPR/Cas9 knockout; TGF-β1, transforming growth factor-beta 1.

FIGURE 2

Conditional screening of BEAS-2B-GeCKO cells identifies 76 top molecular regulators mediating EMT and pulmonary fibrosis. (A) Schematic flow of generating BEAS-2B-GeCKO cells and subsequent TGF-β1 treatment and NGS. (B) A representative image demonstrating the successful transduction of GeCKO v2 sgRNA library into BEAS-2B cells. From the left to right, strips represent the markers, and the detection of GeCKO v2 sgRNA library in BEAS-2B cells, BEAS-2B-NC cells, and BEAS-2B-GeCKO cells, respectively. (C, D) The number of enriched genes represented by 1, 2, 3, 4, 5, or six independent sgRNAs. Genes were identified by total reads of sgRNAs and sgRNA diversity. (E) The Venn diagram analysis of data from our GeCKO screening and GSE104908 confirmed 76 top molecular regulators. EMT, epithelial mesenchymal transformation; GeCKO, Genome-wide CRISPR/Cas9 knockout; sgRNA, single guide RNA; TGF-β1, transforming growth factor-beta 1.

FIGURE 3

Pathway and functional enrichment analysis of top molecular regulators reveals a strong association with EMT and ECM dysregulation. (A) Protein-protein interaction network among the top molecular regulators was constructed in the STRING database. (B) KEGG pathway analysis of the top molecular regulators. Signaling pathways that are closely associated with ECM dysregulation, EMT, and pulmonary fibrosis are highlighted in red. Genes enriched in corresponding pathways are shown below the bars. (C) Through the GO analysis of top molecular regulators, the enriched GO terms indicate the close correlation with ECM. ECM, extracellular matrix; GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K-Akt, phosphatidylinositol 3-kinase-protein kinase B; TGF-β, transforming growth factor-beta.

FIGURE 4

The knockout of candidate genes alters the phenotype of TGF-β1-induced EMT. Ten BEAS-2B cell lines were generated by specifically knocking out each candidate gene, followed by the rTGF-β1 treatment. PBS-treated cell lines served as the control. (A) A heatmap displaying the sgRNA counts of the candidate genes. (B) Upon the knockout of candidate genes, alterations in the mRNA expression of Collagen-I and Fibronectin was determined via RT-qPCR. (C) The effect of knocking out each candidate gene on cell viability was evaluated by CCK-8 assay. (D, E) Compared with control group (a), the effect of knocking out MMP3 (b), COL20A1 (c), COL27A1 (d), COL6A3 (e), COL7A1 (f), SERPINE2 (g), WNT5B (h), SMAD7 (i), INHBA (j), and WNT11 (k) on relative cell migration was assessed using the transwell system (magnification: ×100). Relative cell migration was calculated as the ratio of migrated cells in experimental groups to the background migration in the control group. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA with Tukey’s multiple comparisons test. In (B), n = 6 for the control group; n = 3 for other groups (candidate gene knockout); In (C) and (D), n = 3 per group. * indicates significant difference compared with the control. *P < 0.05, **P < 0.01, ***P < 0.001. NGS, next-generation sequencing; OD, optical density; sgRNA, single guide RNA; TGF-β1, transforming growth factor-beta 1.

FIGURE 5

The knockout of key profibrotic regulators promotes cell apoptosis and reduces ECM components deposition. Ten BEAS-2B cell lines were generated by specifically knocking out each candidate gene, followed by the rTGF-β1 treatment. PBS-treated cell lines served as the control. (A) Representative plots of cell apoptosis for BEAS-2B cell lines. (B) Upon the knockout of candidate genes, the rate of apoptosis for BEAS-2B cell lines was analyzed by flow cytometry. (C) After suppressing the expression of key profibrotic regulators (COL20A1, COL27A1, and WNT11), alterations in the protein expression levels of Fibronectin and Collagen-I were analyzed and quantified by Western blot. Analysis of protein levels in relation to GAPDH. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA with Tukey’s multiple comparisons test (B) or unpaired two-tailed t-test between each condition (C). In (B) and (C), n = 3 per group. * indicates significant difference compared with the control. **P < 0.01, ***P < 0.001. KO, knockout; NC, negative control.

FIGURE 6

Enhanced expression levels of COL27A1 and WNT11 were found in patients with pulmonary fibrosis. (A) The expression level of COL20A1 in GSE24206. (B) The expression level of COL27A1 in GSE24206. (C) The expression level of WNT11 in GSE40839. Data are shown in violin plots, and analyzed using Mann-Whitney U Test. In (A) and (B), n = 6 for the control group; n = 17 for the PF group. In (C), n = 10 for the control group; n = 11 for the PF group. All the violin plots were generated using R software v4.3.3. PF, pulmonary fibrosis.