Transcriptomic Analysis of Genes Associated with Oxidative Stress in Chronic Rhinosinusitis Patients with Nasal Polyps: Identifying Novel Genes Involved in Nasal Polyposis

Abstract

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complicated inflammatory disease, and the underlying mechanism remains unclear. While some reactive oxygen/nitrogen species-related gene products are reported to participate in CRSwNP, a systemic and full analysis of oxidative-stress-associated genes in CRSwNP has not been extensively studied. Therefore, this study sought to catalog the gene-expression patterns related to oxidative stress and antioxidant defense in control and CRSwNP patients. In total, 25 control and 25 CRSwNP patients were recruited. The distribution and expression of 4-hydroxynonenal and 3-nitrotyrosine as markers of oxidative stress-which is represented by lipid peroxidation and the protein nitration of tyrosine residues in CRSwNP nasal polyps (NPs)-were more apparently increased than those found in the control nasal mucosae, as determined by immunohistochemistry (IHC). The expression of 84 oxidative-stress-related genes in nasal mucosae and NP tissues was analyzed via real-time PCR, which showed that 19 genes and 4 genes were significantly up- and downregulated, respectively; among them, inducible nitric oxide synthase (iNOS) and heme oxygenase 1 (HO-1) were notably upregulated, whereas lactoperoxidase (LPO), myeloperoxidase (MPO), and superoxide dismutase 3 (SOD3) were highly downregulated. Changes in the mRNA and protein levels of these redox proteins were confirmed with a customized, real-time PCR array and RT-PCR analysis, as well as Western blotting and IHC assays. A receiver operating characteristic curve analysis further suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors of CRSwNP development. Collectively, we present an oxidative-stress-related gene profile of CRSwNP NP tissues, providing evidence that the systemic changes in oxidative stress and the antioxidative defense system, including novel iNOS, heme peroxidases, and other genes, are closely linked to CRSwNP pathology, development, and progression.

Keywords: CRS; CRSwNP; ROS; lactoperoxidase; oxidative stress; transcriptional profiling.

Figure 1

IHC analysis of 4-HNE and 3-nitrotyrosine expression levels in nasal tissues. The control nasal mucosae and NPs were stained by (A) hematoxylin and eosin (HE) staining and (B) anti-4-HNE Ab or (C) 3-nitrotyrosine Ab and its corresponding nonimmune IgG (NIgG). The selected regions (black rectangle frames (a,b) were magnified and images were captured under a microscope. The images shown below indicate positive staining, in which the deep red color spots/areas indicate the presence of 4-HNE or 3-nitrotyrosination (blue arrows) (panel (a)). A quantitative analysis of similar data for 4-HNE (n = 6) and 3-nitrotyrosine (n = 3) staining is also shown (panel (b)). Scale bar = 100 μm. * p < 0.05 and ** p < 0.01 versus control.

Figure 2

Visualization of fold changes in oxidative stress gene expression between the control and CRSwNP groups for each gene in the array in the context of an array layout. The heat map and table provide fold regulation data used for the map as well as abbreviations for the genes associated with each datum.

Figure 3

Volcano plot of changes in significant oxidative stress gene expressions. The 84 genes in the human nasal mucosae and NPs associated with oxidative stress were analyzed. The volcano plot displays statistical significance versus fold changes on the y- and x-axes, respectively. The x-axis represents fold changes from the control, whereas the y-axis represents the p-values. The vertical solid line in the middle and the other two vertical dashed lines indicate a one-fold change and a threshold of two-fold changes in gene expression, respectively. Note that the light blue line indicates a p-value of 0.05. The significantly changed genes are located outside the two vertical dashed lines and above the blue line, and they are labeled with their respective gene names.

Figure 4

Verifying the expression level of the representative changed genes using RT-PCR analysis. The total RNA was extracted from human control nasal mucosae and CRSwNP NP tissues. (A) The representative downregulated GPX3, SOD3, and MPO and (B) the upregulated NCOA7, NOS2 (iNOS), and HMOX-1 (HO-1) mRNA expression levels were determined using RT-PCR to verify their changes in a real-time PCR array assay. Data are mean ± SEM. C: control; W: CRSwNP. ** p < 0.01 and *** p < 0.001 versus control.

Figure 5

Western blot and IHC analysis of protein expression levels in the representative altered genes in the control nasal mucosae and CRSwNP NPs. (A) The protein levels of the representative altered genes in the control nasal mucosae and CRSwNP NPs, including SOD3, iNOS (NOS2), and HO-1, were analyzed in 18 human nasal control mucosae and 15 CRSwNP NP tissues with Western blotting. The iNOS was analyzed with WB in a nonreduced form. A quantitative analysis of the results was performed using densitometry. Data are mean ± SEM. **** p < 0.0001 versus control. (B) (a) An IHC analysis of SOD1 expression in four representative control nasal mucosae and NP tissues from the control and CRSwNP patients, respectively. Note that the positive staining was mainly found in the epithelium, which is quantified and shown in panel (b) (n = 6). pt: patient; epi: epithelium. Scale bar = 100 μm. ** p < 0.01 versus control.

Figure 6

ROC curve analysis of potential oxidative stress biomarkers used to predict the development of CRSwNP. The markedly changed genes, including LPO, MPO, SOD3, NOS2 (iNOS), and HMOX-1 (HO-1), in the NPs of 25 patients with CRSwNP were analyzed using an ROC curve analysis. The p-values for the genes were 8.32 × 10⁻⁸, 1.06 × 10⁻⁶, 4.22 × 10⁻⁶, 2.24 × 10⁻⁵, and 1.71 × 10⁻⁵, respectively.