Suppression of regulatory factor X 7 alleviates airway remodeling and inflammation in childhood asthma

Abstract

Objective: Childhood asthma is a chronic heterogeneous syndrome composed of distinct disease entities or phenotypes. This study was conducted to characterize regulatory factor X 7 (RFX7) in childhood asthma.

Material and methods: Two available transcriptome datasets (GSE65204 and GSE27011) were used to analyze regulatory factor X (RFX) family members in childhood asthma. Random forest, logistic regression, and linear support vector machine (SVM) analyses were performed to construct an RFX-based classification model. Airway smooth muscle cells (ASMCs) were induced through platelet-derived growth factor-BB (PDGF-BB) for an asthma in vitro model. RFX7 expression was measured through immunoblotting. RFX7 was knocked out by transfection of RFX7 small-interfering RNAs, and then airway remodeling and inflammation were assayed.

Results: Among RFX family members, RFX3, RFX7, and RFX-associated protein displayed differential expression in childhood asthma versus healthy controls. Thus, SVM, logistic regression, and random forest-based machine learning models were built. The random forest model presented the best diagnostic efficacy (area under the curve [AUC] = 1 and 0.67 in discovery and verification sets). RFX7 was found to be effective in diagnosing childhood asthma (AUC = 0.724 and 0.775 in discovery and verification sets). In addition, RFX7 was overexpressed in PDGF-BB-stimulated ASMCs (^{✶ ✶} P < 0.01). Silencing RFX7 remarkably attenuated the proliferative and migrative capacities of ASMCs with PDGF-BB stimulation (^{✶ ✶} P < 0.01). In addition, RFX7 was positively related to neutrophil infiltration in childhood asthma, and its knockdown downregulated the levels of pro-inflammatory cytokines in PDGF-BB-stimulated ASMCs (^{✶ ✶} P < 0.01).

Conclusion: The findings of this study indicate that RFX7 is a novel molecule that is correlated with airway remodeling and inflammation in childhood asthma, providing insights into the mechanism underlying this disease and its potential clinical importance.

Keywords: Airway remodeling; Airway smooth muscle cells; Asthma; Inflammation; Regulatory factor X transcription factors.

Figure 1:

Aberrant transcriptome programs in children with persistent atopic asthma. (a) Selection of genes with differential expression in children with persistent atopic asthma versus healthy controls. Blue, downregulation; red, upregulation; and gray, no significance. (b) Transcriptome patterns of the identified differentially expressed genes (DEGs) in persistent atopic asthma and healthy controls. Blue to red denotes down-to upregulated expression. (c) Transcriptome patterns of the first 20 down-to upregulated genes in persistent atopic asthma in comparison with healthy controls. (d-f) The biological process, cellular component, or molecular function network based on the DEGs. From blue to red means that P-value goes down. The larger the bubble, the more genes are enriched. (g) The kyoto encyclopedia of genes and genomes pathway network based on the DEGs. Machine learning models based on RFX family members for diagnosing childhood asthma. DEGs: Differentially expressed genes, RFX: Regulatory factor X, IGLL5: Immunoglobulin lambda-like polypeptide 5, LOC96610: Hypothetical gene, IGLL1: Immunoglobulin lambda-like polypeptide 1, DEFB4A: Defensin beta 4A, HLA-DOA: Major histocompatibility complex, class II, DO alpha, C4orf7: Follicular dendritic cell-secreted protein, CSF3: Colony-stimulating factor 3, FCGR3A: Fc gamma receptor IIIa, HLADQA1: Major histocompatibility complex, class II, DQ alpha 1, TMIGD2: Transmembrane and immunoglobulin domain containing 2, KLRB1: Killer cell lectin-like receptor B1, TCL1A: TCL1 family AKT coactivator A, CXCL13: C-X-C motif chemokine ligand 13, CXCR6: C-X-C motif chemokine receptor 6, CCL5: C-C motif chemokine ligand 5, C1QB: Complement C1q B chain, TNF: Tumor necrosis factor, FCRL5: Fc receptor-like 5, VNN2: Vanin 2, CST1: Cystatin SN, ITLN1: Intelectin 1, CPA3: Carboxypeptidase A3, TPSD1: Tryptase delta 1, TPSAB1: Tryptase alpha/beta 1, CTSG: Cathepsin G, CCL26: C-C motif chemokine ligand 26, CCK: Cholecystokinin, CAPN14: Calpain 14, KRT6A: Keratin 6A, NTRK2: Neurotrophic receptor tyrosine kinase 2, CDH26: Cadherin 26, ALOX15: Arachidonate 15-lipoxygenase, PRB4: Proline-rich protein BstNI subfamily 4, CEBPE: CCAAT enhancer-binding protein epsilon, SCARNA5: Small Cajal body-specific RNA 5, FAM25A: Family with sequence similarity 25 member A, CYP1B1: Cytochrome P450 family 1 subfamily B member 1, PRB1: Proline-rich protein BstNI subfamily 1, CD36: Cluster of differentiation 36 , HLA-DPB1:major histocompatibility complex, class II, DP beta 1

Figure 2:

Machine learning models based on the regulatory factor X (RFX) family members for diagnosing childhood asthma. (a) The single– sample gene set enrichment approach (ssGSEA) score of the RFX family members in childhood asthma and healthy controls. Blue to red represents low-to-high ssGSEA score. (b) Comparison of the RFX ssGSEA score in childhood asthma versus healthy controls. ^✶✶✶P < 0.001. (c-e) Receiver operating characteristics for the evaluation of the diagnostic efficacy of linear support vector machine, logistic regression, and random forest–based machine learning models based on the RFX family members in the discovery and verification sets. (f-h) Assessment of the performance of RFX3, RFX7, and RFX-associated protein in diagnosing childhood asthma in the two sets.

Figure 3:

Regulatory factor X (RFX7) displays the notable overexpression in asthma cell models. (a) Representative Western blotting images of RFX7 in airway smooth muscle cells (ASMCs) administrated with 50 ng/mL platelet-derived growth factor-BB (PDGF-BB) or control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as a reference control. (b) Quantification analysis of RFX7 expression in control or PDGF-BB-induced ASMCs. (c) Representative Western blotting images of RFX7 in ASMCs in the context of small interfering-negative control (si- NC), RFX7 Small interfering RNAs (siRNA)#1, RFX7 siRNA#2, or RFX7 siRNA#3 transfection. (d) Quantification analysis of RFX7 expression in si-NC, RFX7 siRNA#1-, RFX7 siRNA#2-, or RFX7 siRNA#3-transfected ASMCs. (e) Representative reverse transcription quantitative polymerase chain reaction of RFX7 in ASMCs in the context of si-NC, RFX7 siRNA#1, RFX7 siRNA#2, or RFX7 siRNA#3 transfection. ^✶✶ compared with the control group at P < 0.01. The experiment was repeated 3 times, n = 3.

Figure 4:

Regulatory factor X7 (RFX7) knockdown alleviates platelet-derived growth factor-BB (PDGF-BB)-triggered airway smooth muscle cell (ASMC) proliferation. (a) Representative images of 5-ethyny-2'-deoxyuridine (EdU) assay in ASMCs in the context of PDGF-BB administration or RFX7 Small interfering RNAs (siRNA) transfection. Scale bar, 50 μm. (b) Quantification analysis of EdU-positive ASMCs with PDGF-BB administration or RFX7 siRNA transfection. ^✶✶P < 0.01. The experiment was repeated 3 times, n = 3.

Figure 5:

Targeting regulatory factor X7 (RFX7) ameliorates airway smooth muscle cell (ASMC) migration stimulated by platelet-derived growth factor-BB (PDGF-BB). (a) Representative photographs of wound healing assay in ASMCs with PDGF-BB stimulation or RFX7 small interfering RNAs (siRNA) transfection. Scale bar, 100 μm. (b) Quantification of the closure ratios of ASMCs with regard to PDGFBB treatment or RFX7 siRNA transfection. (c) Representative photographs of Transwell assay in ASMCs stimulated with PDGF-BB or transfected with RFX7 siRNAs. Cell mobility = (initial scratch width − final scratch width)/initial scratch width × 100%. (d) Quantification of migrative ASMCs with regard to PDGF-BB administration or transfection with RFX7 siRNAs. Scale bar, 50 μm.; ^✶✶P < 0.01. The experiment was repeated 3 times, n = 3.

Figure 6:

Silencing regulatory factor X7 (RFX7) weakens platelet-derived growth factor-BB (PDGF-BB)-stimulated airway smooth muscle cell (ASMC) remodeling. (a) Representative photographs of immunofluorescence on alpha smooth muscle actin (α-SMA) in ASMCs with PDGFBB stimulation or RFX7 small interfering RNAs (siRNA) transfection. Scale bar, 150 μm. (b) Quantification analysis on α-SMA in ASMCs with PDGF-BB stimulation or RFX7 siRNA transfection. (c) Representative photographs of immunofluorescence on N-cadherin in ASMCs administrated with PDGF-BB or transfected with RFX7 siRNAs. Scale bar, 150 μm. (d) Quantification analysis on N-cadherin in ASMCs administrated with PDGF-BB or transfected with RFX7 siRNAs. (e) Representative photographs of immunofluorescence on E-cadherin in ASMCs with regard to PDGF-BB stimulation or RFX7 siRNA transfection. Scale bar, 50 μm. (f) Quantification analysis on E-cadherin in ASMCs with regard to PDGF-BB stimulation or RFX7 siRNA transfection. ^✶✶P < 0.01. The experiment was repeated 3 times, n = 3.

Figure 7:

Targeting regulatory factor X7 (RFX7) decreases inflammatory response in platelet-derived growth factor-BB (PDGF-BB)-stimulated ASMCs. (a) Correlation analysis on the RFX single–sample gene set enrichment approach score, RFX7, and neutrophils in childhood asthma. (b-d) reverse transcription quantitative polymerase chain reaction on the detection of mRNA levels of tumor necrosis factor alpha-like, interleukin 1 beta, and interleukin 6in ASMCs with PDGF-BB stimulation or transfection with RFX7 siRNAs. ^✶✶P < 0.01. The experiment was repeated 3 times, n = 3.