Introduction
Psoriasis is an inherent and inflammatory skin disease with scaly erythematous lesions, affecting 2–3% of the Caucasian population (1). The scales are a result of a hyperproliferative epidermis, as well as parakeratosis, and the highly mitotic rate of psoriatic keratinocytes results in a thickened epidermis. Complex pathogenic mechanisms associated with genetic expression variances and different immune responses result in psoriasis aggravation (2).
In addition to hyperproliferation, the psoriatic keratinocytes exhibit an abnormal resistance to apoptosis. Deceleration in keratinocyte apoptosis is the most significant pathological change in psoriasis (3). The decrease in apoptosis, which causes imbalances in skin homeostasis, may be associated with the induction of psoriatic hyperplasia (3). Multiple factors have been identified to contribute to anti-apoptosis in psoriatic keratinocytes, including the activation of mitogenic signaling pathways and the inactivation of certain apoptotic molecules (4,5). In the present study, various PageRank hub genes were identified to be associated with anti-apoptosis, as well as with the regulation of apoptosis in psoriasis. Furthermore, the current study identified that the target genes in anti-apoptosis and apoptosis regulation are tightly associated with the ESR1 gene. The ESR1 gene encodes an estrogen receptor α (ERα). The estrogen stimulates a rapid relocation of ERα to induce high mitochondrial gene expression (6). It has been clearly demonstrated that activation of the mitochondrial signaling pathway, as well as the extrinsic apoptosis signaling pathway, is key in the process of many forms of programmed cell death (5). Thus, it was hypothesized in the present study that ERα may exert a protective role against psoriatic proliferation.
The cytokine related Toll-like receptor (TLR) signaling pathway, a classical signaling pathway, is considered to be important in the progression of psoriasis (7). However, based on the analyses of the present study, the pathways in cancer was the most significant pathway that correlated with psoriasis, demonstrating a more significant association than the TLR signaling pathway. Pathways in cancer affect tumor proliferation by protecting the tumor from cytokine-induced apoptosis. The current results suggest that the activation of pathways in cancer may inhibit psoriatic keratinocyte apoptosis.
To analyze the disease-associated genes and gene functions, the differentially expressed genes (DEGs) associated with psoriasis were screened and compared with healthy controls from the DNA microarray database. A gene interaction network was generated for topological analysis of relevant genes associated with psoriasis using PageRank and the gene enrichment functions were subsequently evaluated. The top-ranked PageRank hub gene, ESR1, which is down-regulated in psoriasis, exhibited binding sites enriched with anti-apoptotic genes that provides support for the negative association between estrogen and apoptosis. The current results demonstrate that reduced levels of ERα expression result in the anti-apoptotic behavior of psoriatic keratinocytes by activating anti-apoptotic gene expression. Pathways in cancer may protect the psoriatic cell from apoptosis by inhibiting ESR1 expression. The present study provides evidence regarding ESR1 gene function and demonstrates that the interaction with anti-apoptotic genes is involved in the underlying biological mechanisms of apoptosis resistance in psoriasis.
Materials and methods
Affymetrix microarray data and preprocessing
The microarray gene expression profiles were extracted from the Gene Expression Omnibus (GEO) database (GEO accession no. GSE13355; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13355) (8,9), which contained gene expression profiles for 58 psoriatic patients and 64 healthy control subjects. All samples were run on GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), microarrays containing >54,000 gene probes. The raw data were processed using the Robust Multichip Average (RMA) method (5). The expression values were adjusted using RMA expression values (on the log scale) to account for batch and gender effects.
DEG identification
DEGs between psoriasis and normal skin were identified using the limma R package (10). The raw P-value was corrected by the Benjamin and Hochberg method to circumvent the multi-test bias (11). Finally, the false discovery rate (FDR) <0.01 and |log fold change (FC)| >1 were used as the cut-off for DEG identification.
Interaction network construction of DEGs
A gene interaction network was generated by mapping all the DEGs in the Search Tool for the Retrieval of Interacting Genes (STRING) database, which is a database for predicting functional associations between proteins (12). The interactions include direct (physical) and indirect (functional) associations, and all of the associations are derived from four sources: Genomic context, high-throughput experiments, coexpression (conserved) and previous knowledge (13). All of the associating pairs were selected based on a combined probabilistic confidence score.
Identification of top-ranked hub genes using Google PageRank score
In order to identify hub genes (key genes), the PageRank score was calculated in all DEGs. The PageRank analysis was initially applied to Google (the web search engine) to identifying significant web pages (14–16) and has been used during the robust analysis of protein networks to identify important nodes (17). Unlike simply calculating the degree of each gene, the PageRank score measures the importance or popularity of a gene based solely on the interaction (link) structure of the interaction network. It selects the genes that exhibit a high degree, whilst also maintaining the important low-degree genes, which link to other important genes in the protein-protein interaction network. It is expected that the genes with more interaction links, especially those from important genes, are given higher PageRank scores.
Gene-annotation enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (David)
DAVID software (Laboratory of Human Retrovirology and Immunoinformatics, Clinical Services Program; Leidos Biomedical Research, Inc., Frederick, MD, USA) (18,19), a gene-annotation enrichment analysis tool, adopts a common core strategy to systematically map a hubs gene list to the associated biological annotation [for example, gene ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways], and then statistically highlight the most overrepresented (enriched) biological annotation out of thousands of linked terms and contents. In the present analysis, GO and KEGG pathway annotations were integrated to evaluate the function enrichment of the hub genes.
Statistical analysis
DAVID software (18,19) was used for all statistical analyses, and GO terms and KEGG pathways were selected using a standard parameter setting (FDR<0.05 and P<0.1). All the known human genes were used as the background for Fisher's exact test, and a P-value was calculated and the Bonferroni correction was applied.
Results
DEG analysis
After extracting the gene expression data from the public dataset, GSE13355, the DEGs between psoriasis lesions and normal skin were identified. DEGs (n=927; 449 upregulated and 478 downregulated) were selected (FDR <0.01 and |logFC| >1) and a heat map of the DEG expression level between the two groups is presented in Fig. 1.
Construction and topological analysis of an interaction network
The interaction network was constructed using STRING and the DEGs that had been identified. In total, 640 genes were identified and the interaction network of the DEGs is presented in Fig. 2. Based on the network topological analysis, it was found that the protein interaction network exhibited scale-free attributes. Three network topologies were processed using statistical analyses and are presented in Fig. 3. Fig. 3A shows the node degree distribution in the network, the x-axis represents the node degrees (the number of direct connections the node has with other nodes) and the y-axis represents the number of nodes with the different degrees. The node degree distribution indicates that both loosely and closely connected nodes exist in the network. Fig. 3B demonstrates the clustering coefficient distribution in the network, and the clustering coefficient displays the aggregation degree of nodes; the distribution range of the clustering coefficient is between 0 and 1. Fig. 3C shows the path length of the network, indicating that the predominant path length was ~3–4. Subsequent to the analysis of the three topological property parameters of the interaction network, the present study was able to demonstrate the small-world effect of the network, which is subject to scale-free property (20).
PageRank hub genes and enrichment analysis
PageRank analysis was adopted to select the most popular and important genes within the interaction network. A high PageRank score is associated with a high degree. According to analysis of the PageRank score, 14 genes with the highest PageRank scores were selected as the top hub genes (Table I). Thirteen of the top 14 hub genes are highlighted in red and ESR1, identified as the top hub gene, was highlighted in yellow in the interaction network (Fig. 2). ESR1, representing high relativity with psoriasis, demonstrates a downregulated level of expression in Table I. The other 13 of the top hub genes, demonstrating various levels of expression, are presented in Table I.
To gain further insight into the functions of the top 14 hub genes, the enrichment of GO functions and KEGG pathway analysis were performed using the DAVID tool. The significant GO enrichment terms are presented in Table II. The function of anti-apoptosis was confirmed to be highly associated with psoriasis. Six of the top hub genes [ESR1, apolipoprotein E (APOE), myeloid cell leukemia-1 (MCL1), cartilage oligomeric matrix protein (COMP), baculoviral IAP repeat containing-5 (BIRC5) and interleukin-1β (IL-1B)] were identified to be involved in the anti-apoptosis effects of psoriasis (Table II). The other functions, such as regulation of apoptosis, programmed cell death and cell death, also represent the association with psoriasis. The same eight top hub genes [ESR1, APOE, MCL1, COMP, BIRC5, IL-1B, matrix metalloproteinase-9 (MMP9) and signal transducer and activator of transcription-1 (STAT1)] were observed to be correlated with regulation of apoptosis, programmed cell death and cell death (Table II).
In addition, the ESR1 topological neighbors were selected from the interaction network; 56 pairs that directly interacted with the ESR1 gene are shown in Fig. 4, including nine top-ranked hub genes [APOE, BIRC5, cyclin D1 (CCDN1), FBJ murine osteosarcoma viral oncogene homolog (FOS), interleukin-8 (IL-8), lipoprotein lipase (LPL), MMP9, STAT1, signal transducer and activator of transcription-3 (STAT3)] highlighted in red. The five genes (APOE, MCL1, COMP, BIRC5 and IL-1B) with anti-apoptotic function were directly or indirectly associated with the ESR1 gene (Fig. 4). Three genes were indirectly linked with the ESR1 gene by binding to the ESR1 gene topological neigbors, particularly by binding to the important apoptosis-regulating genes, such as B-cell CLL/lymphoma 2 (BCL2), MMP9 and CCND1.
Using KEGG pathway analysis (FDR <0.05), the pathways in cancer was detected to be most significantly associated with psoriasis, more so than the TLR signaling pathway, as shown in Fig. 5. Other pathways, including the chemokine signaling pathway and Janus kinase/signal transducers and activators of transcription signaling pathway were also detected in the present study; however, the significance was identified to be relatively lower. Seven of the top hub genes (FOS, CCND1, IL-8, MMP9, BIRC5, STAT1 and STAT3) were detected in response to pathways in cancer and five of the top hub genes [FOS, IL-8, IL-1B, STAT1 and chemokine (C-X-C motif) ligand 10 (CXCL10)] exerted an effect on the TLR signaling pathway (data not shown). All of the above-mentioned top hub genes, apart from the CXCL10 gene, were involved in the first two pathways, demonstrating a direct association with the ESR1 gene (Fig. 4). The present study demonstrated that four of the genes (MMP9, BIRC5, IL-1B and STAT1) were highly associated with anti-apoptosis, regulation of apoptosis and programmed cell death.
Discussion
Psoriasis is a chronic inflammatory skin disorder. Significant decreases in keratinocyte apoptosis is a specific pathogenic phenomenon in psoriasis lesions compared with normal skin (3). In the present study, 927 DEGs were identified from the GEO database, GSE13355, which contained gene expression profiles for 58 psoriatic lesion samples and 64 normal skin samples. An interaction network with the 927 DEGs was then constructed (Fig. 2). Following PageRank analysis (Table I), ESR1 and another 13 genes were selected as the top hub genes from 640 DEGs according to their PageRank score. In addition, the functions of the 14 top hub genes were analyzed by using GO term function analysis (Table II), indicating that the most significant enrichment function was associated with anti-apoptosis. Other functions, including response to organic substances, and regulation of apoptosis, programmed cell death and cell death, were also highly associated with psoriasis.
Apoptosis is a type of programmed cell death. Multiple factors control the extrinsic and intrinsic apoptotic signaling pathways. To maintain epidermal homeostasis, the complex apoptotic process is regulated by numerous anti-apoptotic molecules (5). The ESR1 gene, as well as five other top hub genes, has been demonstrated to exert an effect on anti-apoptosis in psoriasis according to the present results. Four of the genes (MCL1, COMP, BIRC5 and IL-1B) demonstrate an upregulated expression level in psoriasis. The other two genes (ESR1 and APOE) demonstrate a downregulated level of expression. In addition to the six top hub genes, a further two highly expressed genes, MMP9 and STAT1, were identified to be involved in the regulation of apoptosis, programmed cell death and cell death. The MCL1 gene encodes an anti-apoptotic protein, myeloid cell leukemia-1. Ultraviolet B radiation therapy inhibits growth and induces apoptosis of human skin cells by downregulating MCL1 expression (21). IL-1B induced IL-8 and p53 mRNA expression, as well as protein production of IL-8 (22). p53 overexpression has been described as a physiological reaction to hyperproliferation in order to resist keratinocyte apoptosis. Furthermore, knockdown of p53 expression levels in human keratinocytes accelerates MCL1 reduction, thereby enhancing apoptosis (23).
The ESR1 gene encodes an ERα (24), which is a ligand-activated transcription factor that binds to estrogen (25). ERα is the receptor that transmits estrogen signaling; however, the function of ERα in psoriasis development remains largely unknown. In the present study, the anti-apoptotic genes (BIRC5 and APOE) as topological neighbors, demonstrated a direct association with the ESR1 gene (Fig. 4). Other anti-apoptotic genes (MCL1, IL-1B and COMP) demonstrated indirect connections with the ESR1 gene by binding to the ESR1 topological neighbors, particularly by binding to the important apoptosis regulation genes, such as BCL-2, MMP9 and CCND1 (Fig. 4). The MMP9 gene, an important gene in apoptosis regulation, has been demonstrated to be involved in the cell death pathways (26). Psoriasin (termed, S100 calcium-binding protein A7), which is highly expressed in tumors and hyperproliferative skin, enhances anti-apoptotic nuclear factor-κB (NF-κB)-mediated MMP-9 secretion in ERα− breast cancer cells, but has the opposite effect in ERα+ cells (27,28). CCDN1 and BCL2, at lower expression levels, were also associated with the ESR1 gene (Fig. 4; Table I). A previous study regarding colorectal cancer demonstrated that 17β-estradiol activates p53 to inhibit cell proliferation by inhibiting CCDN1 (29). In addition, various studies have reported that a lower expression level of B-cell lymphoma 2 (Bcl-2) was strongly associated with psoriasis (4,30–32). The strong bcl-2-like protein 4 (BAX) expression, as well as the concomitant decrease in the level of Bcl-2 expression indicated a resistance to BAX-mediated apoptosis in psoriasis (30). Thus, the reduced expression level of ESR1 may lead to the upregulated expression of anti-apoptotic genes via mediation of the genes that are associated with apoptosis regulation, such as MMP9, BCL2 and CCND1. This provides evidence that the ESR1 gene may be implicated in psoriatic apoptotic resistance and that ERα may exert a protective role against psoriatic proliferation. To the best of our knowledge, the underlying mechanism of the association between ESR1 and the anti-apoptosis signaling pathway in psoriasis has not been fully elucidated; however, the present findings indicate that the reduced level of ESR1 gene expression associated with psoriasis, is highly correlated with apoptosis resistance.
Using KEGG pathway analysis for the top 14 hub genes, the signaling pathway most significantly linked with psoriasis is the pathways in cancer, as well as the TLR signaling pathway (Fig. 5). As a classical pathway in psoriasis, the TRL signaling pathway has been shown to be important in psoriasis and is involved in detecting invading micro-organisms and producing pro-inflammatory stimuli to the skin (7). TLRs drive aberrant activation and unrestricted inflammatory responses (33).
In the current study, it is the first time, to the best of our knowledge, that the pathways in cancer has been detected to have the most significant association with psoriasis, more so than the TLR signaling pathway. Pathways in cancer exerts an effect on tumor proliferation by protecting tumor cells from cytokine-induced apoptosis. A previous study demonstrated that the thickened psoriatic skin displays a particular resistance of keratinocytes to interferon-γ (INF-γ)- and tumor necrosis factor-α (TNF-α)-induced apoptosis by sustaining the activation of the phosphatidylinositol 3-kinase/Akt signaling pathway and consequently activating the anti-apoptotic NF-κB cascade (4). Thus, the mechanisms of cell proliferation result in activation of anti-apoptotic molecules, which contribute to suppressing cytokine-induced apoptosis and maintaining the response to pro-inflammatory stimuli in keratinocytes (34). Hsu et al (35) reported that ERα inhibited bladder cancer cell growth by inhibiting Akt activity (35). Therefore, the present study hypothesized that ERα expression may activate INF-γ and TNF-α-induced apoptosis in cells by inhibiting Akt activity in psoriasis and various types of cancer, including bladder cancer.
The current study demonstrated that seven of the top hub genes that were involved in the pathways in cancer were directly associated with the ESR1 gene. The above-mentioned evidence suggests that activating the pathways in cancer may protect the psoriatic cell from cytokine-induced apoptosis by inhibiting ERα expression. Further investigations are required to establish why the pathways in cancer is more significant than the cytokine-related TLR signaling pathway in apoptosis resistance in psoriasis.
In conclusion, activating the pathways in cancer, as well as reducing ERα expression promotes proliferation and resistance to apoptosis. The present results indicate that a lower ERα expression level may induce psoriatic keratinocyte proliferation and anti-apoptosis effects via anti-apoptotic genes modulated by activation of the pathways in cancer. However, the contribution of individual factors requires elucidation. Future studies are required to investigate the functions of ERα, the association between anti-apoptosis genes in psoriasis and whether these genes are involved in the biological mechanisms underlying the coordination of psoriatic keratinocyte apoptosis resistance.