Analysis of the expression and potential molecular mechanism of interleukin-1 receptor antagonist (IL1RN) in papillary thyroid cancer via bioinformatics methods

Abstract

1 Introduction

Thyroid carcinoma (THCA) is the most common endocrine malignancy worldwide [1]. The incidence of THCA has increased sharply over the past 3 decades [2]. Papillary thyroid carcinoma (PTC) is the major subtype of THCA, accounting for more than 90% of cases [3]. The clinical course of PTC is generally indolent, and the cancer-specific mortality of PTC is low compared to that of other cancers [4]. However, the incidence of cervical lymph node metastasis is high, which leads to local recurrence and poor prognosis [5]. The main task for the future is to identify high-risk patients and to give them appropriate treatment and care [6]. Therefore, further studies are required to explore the underlying mechanisms of tumorigenesis as well as identify additional biomarkers that predict prognosis and serve as therapeutic targets.

The interleukin-1 (IL-1) family of cytokines are the most effective molecules in the innate immune system [7]. IL-1 receptor antagonist (IL1RN) was initially found as a natural antagonist of IL-1[8]. IL1RN is structurally related to IL-lα and IL-lβ but binds to IL-1 receptors on various target cells without inducing any discernible biological responses [9]. The balance between IL1RN and IL-1 plays a crucial role in many diseases, including cancer [10]. IL1RN has been studied in several cancers, including prostate carcinoma [11–12], cervical carcinoma [13], gastric carcinoma [14], bronchogenic carcinoma [15], endometrial cancer [16], lung cancer [17], ovarian cancer [18], oral malignancies [19], leukemia [20], and other cancers.

Two structural variants of IL1RN have been described: the soluble extracellular form (sIL-1ra) and the intracellular form (icIL-1ra) [4]. Niedzwiecki, S. and colleagues assayed the serum levels of IL1RN in thyroid cancer patients. They measured preoperative IL1RN serum levels of patients with thyroid cancer, and the results showed that the serum concentrations of sIL-1ra were significantly higher in anaplastic carcinoma (ATC) and follicular carcinoma (FTC) patients [21]. To the best of our knowledge, no reports have been published to date concerning IL1RN expression in thyroid tissue. The objective of this study was to investigate the expression of IL1RN in normal and papillary thyroid cancer tissues by performing bioinformatics analysis to elucidate its possible role in tumor progression.

2 Materials And Methods

3 Results

3.1 The differential expression and diagnostic value of IL1RN

The expression levels of IL1RN were initially analyzed in the TCGA cohort consisting of 512 PTC samples and 58 normal samples (Figure 1A). Furthermore, expression data from four independent PTC cohorts obtained from the Gene Expression Omnibus (GEO) (GSE3467, GSE33630, GSE58545, and GSE60542) were employed for validation (Figure 5A-D). Both the GEO and TCGA patient cohorts had significantly higher IL1RN mRNA expression in PTC tissues than in normal tissues (P<0.05). Elevated levels of IL1RN protein expression were observed in PTC tissues compared with those in normal tissues by immunohistochemistry performed using the IL1RN antibody CAB9633. (Figure 1C)

ROC curves were constructed to evaluate the diagnostic value of IL1RN for PTC. The area under the ROC curve of the TCGA cohort was 0.7084 (Figure 1B) and that of each GEO cohort was 0.9753, 0.7279, 0.8868, and 0.8384, respectively. (Figure 5E-H) These results suggest the good positive diagnostic value of IL1RN for PTC.

3.2 Prognostic value and clinical significance of IL1RN

The PTC samples from the TCGA cohort were divided into two groups according to the IL1RN expression level: the high-expression group (H-IL1RN) (n = 116) and the low-expression group (L-IL1RN) (n = 385). Differences in progression-free survival (PFS) between the two groups were analyzed by Kaplan-Meier analysis. (Figure 1D) The results showed that patients with high IL1RN expression had significantly shorter PFS than those with low expression (P=0.0034). However, the data for the multivariate analysis by the Cox regression model suggested that IL1RN expression was not a significant independent prognostic risk indicator for PTC (hazard ratio, 1.033; 95% confidence interval, 0.999−1.068, p > 0.05) (Table 1). Age was an independent predictor of poor prognosis (P＜0.05).

To examine the clinical significance of IL1RN in PTC, the relationship between the expression level of IL1RN and clinical characteristics was investigated by the chi-square test (Table 2). The results suggested that IL1RN expression was significantly associated with age, clinical stage, N stage, T stage, pathologic type, BRAF mutations and RAS mutations.

3.3 Gene coexpression and pathway enrichment analysis

We identified the genes that are significantly positively or negatively related to IL1RN. The result is presented as a volcano plot (Figure 2A). Red and green colors indicate a positive and negative correlation, respectively. The top 50 positively (Figure 2B) or negatively (Figure 2C) correlated genes are depicted by heatmaps. In addition, 2 hub genes (CCL20 and FN1) were identified from the protein–protein interaction (PPI) network (Figure 2D).

GO enrichment analysis revealed that the identified genes were mainly enriched for the GO biological process terms “neutrophil mediated immunity”, “positive regulation of cytokine production”, “adaptive immune response” and “immune response-regulating signalling pathway” (Figure 2E). Further KEGG enrichment analysis suggested that the identified genes were mainly involved in pathways associated with “cell adhesion molecules (CAMs)”, “cytokine-cytokine receptor interaction”, and “phagosome” (Figure 2F).

3.4 Immune-related analysis of IL1RN

Using ESTIMATE, the association between IL1RN expression and immune infiltrates was analyzed. We found that the H-IL1RN group showed a higher immune score (Figure 3B) and ESTIMATE score (Figure 3C) and lower tumor purity (Figure 3D) than the L-IL1RN group. No significant difference in the stromal score was found between the two groups (Figure 3A). The correlations between the IL1RN expression level and the infiltration of six immune cell types in PTC were estimated. The results demonstrated that the expression of IL1RN was found to be significantly correlated with the infiltration of immune cells, especially neutrophils and dendritic cells (Figure 3E). Correlations between IL1RN expression and 28 TIL types (Figure 3F), chemokines (Figure 3G), immune-activating cytokines (Figure S2A), immunosuppressive cytokines (Figure S2B), MHC molecules (Figure S2C), and chemokine receptors (S2D) in pancancer are shown in heatmaps. The correlation analysis results showed a good correlation between IL1RN expression and immune-related molecules.

3.5 Methylation-related analysis

The data showed that the degree of IL1RN DNA methylation was higher in carcinoma tissues than in normal tissues (P<0.0001) (Figure 4A). To further determine the role of methylation, we performed a correlation analysis between the DNA methylation levels of IL1RN and the expression levels of IL1RN. As expected, DNA methylation was negatively correlated with IL1RN expression (R=-0.600, P=2.29e-50) (Figure 4B). Tumor purity was significantly positively associated with IL1RN methylation (R=0.447, P=7.49e-32) (Figure 4C). Patients with an advanced clinical stage (Figure 4D), advanced T stage (Figure 4E) and lymph node metastasis (Figure 4F) tended to have decreased IL1RN methylation levels. In the pathological subtype of PTC, the tall cell type variant of PTC had the lowest degree of methylation, followed by the classical type, and the follicular variant of PTC had the highest degree of methylation (Figure 4G). To further explore the associated functional changes in IL1RN methylation, GO (Figure 4I) and KEGG (Figure 4F) enrichment analyses were performed. The results showed that methylated IL1RN had the opposite effect of IL1RN. Furthermore, the most significant methylation sites are shown in Table 3.

3.6 Pancancer analysis of IL1RN

We analyzed the IL1RN expression levels in different kinds of tumors via the GEPIA platform (Figure 6A). The results showed that the expression levels of IL1RN varied greatly in different cancer types. Of the 33 cancer types tested, 12 cancer types were associated with significantly increased IL1RN expression. Subsequently, the relationship between IL1RN and OS in pancancer was investigated (Figure 6B). Kaplan-Meier survival curves for the high IL1RN expression group (4745 patients) and low IL1RN expression group (4750 patients) indicated that increased IL1RN expression was associated with a shorter survival time in pancancer (HR=1.6, P<0.0001) (Figure 6C). The results showed that increased IL1RN expression levels were significantly associated with shorter OS in kidney renal clear cell carcinoma (KIRC) (Figure 6D), brain lower grade glioma (LGG) (Figure 6E), pancreatic adenocarcinoma (PADD) (Figure 6F) and uterine corpus endometrial carcinoma (UCEC) (Figure 6G).

3.7 Related mutant gene analysis

The analysis of the relationship between IL1RN expression and PTC mutations was performed by linkedOmics (Figure S1A). The IL1RN expression level was further compared between the groups with wild-type and mutant variants of BRAF, NRAS and HRAS (Figure S1B-D). The expression of IL1RN was significantly higher in PTC with the BRAF mutant than in PTC with the wild type (Figure S1B). However, mutated NRAS (Figure S1C) and mutated HRAS (Figure S1D) were correlated with decreased IL1RN expression.

4 Discussion

In this study, we first revealed high IL1RN expression in PTC tissues and its diagnostic and prognostic value by integrated bioinformatics analysis. To further explore the potential function of IL1RN, we identified genes related to IL1RN expression, and functional enrichment analyses were conducted. The results of the KEGG pathway and GO analyses revealed the significant enrichment of genes in immune-related pathways. Our work further demonstrated that high IL1RN expression was significantly associated with decreased tumor purity and increased immune infiltration. Through methylation-related analysis, we determined that DNA methylation might be a regulatory mechanism of IL1RN. In addition, the analysis of the relationship between IL1RN expression and PTC mutations showed that high IL1RN expression was associated with mutated BRAF, wild-type NRAS and wild-type HRAS. Finally, IL1RN shows superior prognostic value for various cancers according to the pancancer analysis.

Niedzwiecki et al [21] revealed that the serum levels of IL1RN were upregulated in patients with ATC and PTC, but no statistically significant difference was found in PTC. We found that the expression of IL1RN was increased in PTC tissues compared with that in normal tissues in 1 TCGA cohort and 4 GEO cohorts and suggested that IL1RN might be used as a potential diagnostic biomarker in PTC. Our results were consistent with those of previous reports on the upregulation of IL1RN in cervical carcinoma [13], gastric cancer [14], lung cancer [15], and endometrial cancer tissues [16]. These findings are also basically consistent with the results of our pancancer study. In contrast to these findings, IL1RN exhibited low expression in oral squamous cell carcinomas (OSCCs) [19].

The results of this study showed the value of IL1RN as a clinical biomarker in PTC and emphasized its potential as a prognostic biomarker in PTC patients. PTC patients with high IL1RN expression had decreased PFS compared to those with low IL1RN expression. Furthermore, high expression of IL1RN was significantly correlated with clinical stage, lymph node metastasis and pathological type. Various studies have highlighted the significant association between IL1RN expression and poor cancer prognosis; however, the results of these studies are conflicting. Van Le et al. [16] revealed that there was a significant correlation between the stage of endometrioid carcinoma and the expression of IL1RN. In a study of colorectal cancer, serum IL1RN levels were correlated with tumor size, local and distant metastasis and vascular infiltration [32]. An increase in IL1RN expression was correlated significantly with lymph node and liver metastases in gastric carcinoma [14]. In contrast, low levels of IL1RN have been associated with increased disease severity in myeloma [22], colorectal cancer [23] and prostate cancer [24].

The underlying mechanism of IL1RN in cancer development and progression is complicated and unclear. In this study, functional enrichment analysis of genes coexpressed with IL1RN showed that IL1RN participates in immune-related biological processes. Consistent with our findings, previous studies have focused on the role of IL1RN in tumor immunity. A study of gastric carcinoma [14] pointed out that on the one hand, IL1RN may promote tumor growth via the impairment of cellular immunity; on the other hand, IL1RN enables malignant cells to escape host immune responses. Smith, D. R. et al. [15] revealed that increased IL1RN in bronchogenic carcinoma is not accompanied by increased IL-1β activity. The altered balance of IL1RN and IL-1β may result in impaired immune surveillance and cytotoxic activity. An experimental study of human glioblastoma cells showed that IL1RN secreted by tumor cells can counteract IL-1 function, which represents a potential escape mechanism that supports cancer growth [31]. In our study, IL1RN was significantly positively correlated with lymph node metastasis and tumor stage, so we speculated that IL1RN might also promote tumor aggressiveness and poor prognosis through immune-related mechanisms in PTC.

IL1RN is an endogenous natural antagonist of IL-1[8], so the discovery of the interaction between IL1RN and IL-1 family molecules is a breakthrough in exploring the function of IL1RN. ONOZAKI et al. [25] reported that IL-1 is a cytocidal factor against several tumor cell lines. IL-1 may enhance cytotoxic T cell activity [29], the tumoricidal capacities of natural killer (NK) cells [30], and monocyte-mediated cytotoxicity [37]. Because IL-1 is critical for tumor immunity, elevation of IL1RN expression may result in a general environment favourable to tumor cells and enhance the metastatic and recurrence potential of tumors by changing local IL-1-dependent pathways.

IL-1β has been reported as an anticancer factor that acts to suppress proliferation and reduce the invasive potential of human PTC cells [26]. sIL-1ra has been shown to block IL-1 function by binding to IL-1 receptors at the cell membrane level [27]. IcIL-1ra is postulated to inhibit intracellular IL-1 activity [28]. Therefore, whether IL1RN can block the function of IL-1 and inhibit the anticancer effect of IL-1β in PTC is worth further study.

In our study, IL1RN showed a significant positive correlation with immune cells, which may be because a variety of immune cells can produce IL1RN through the stimulation of cytokines. Neutrophils produce IL1RN in response to granulocyte/macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) [34]. IL-4 and IL-10 have been reported to increase the production of IL1RN by human monocytes [35]. Yanagawa, H.et, al. reported that IL-13 increases IL1RN production by human alveolar macrophages [36]. It was reported that the apoptotic cell death of monocytes was enhanced by administration of recombinant IL1RN [33]. Our study also showed a significant correlation between IL1RN expression and various cytokines, which implied that IL1RN may play a complex role in tumor immunity.

Our study suggests that methylation is a pretranscriptional regulatory mechanism for IL1RN. The methylation level of IL1RN was negatively correlated with its expression level and was also correlated with the disease stage, lymph node metastasis, and pathological type.

IL1RN is likely to be a potential biomarker associated with the diagnosis and prognosis of PTC. However, although the diagnostic value, prognostic value and molecular functions of IL1RN in PTC have been analyzed through bioinformatics methods, the conclusions have not yet been confirmed by experiments. Therefore, further research is necessary to explore the role of IL1RN in PTC and the pharmacological value of IL1RN as a therapeutic target.

5 Conclusions

IL1RN is a good prognostic and diagnostic biomarker of PTC. IL1RN may promote thyroid cancer progression through immune-related pathways. Methylation may act as an upstream regulator of IL1RN expression and biological function. Additionally, IL1RN showed broad prognostic value in an analysis of a pancancer cohort.

Abbreviations

Declarations

1 Ethics approval and consent to participate

Not applicable

2 Consent for publication

Not applicable

3 Availability of data and materials

The datasets analysed during the current study are available in The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/) or the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/).

4 Competing interests

The authors declare that they have no competing interests.

5 Funding

This research was supported by National Natural Science Foundation of China (81600602).

6 Authors’ contributions

ZX designed the analytical strategies, performed data analyses and wrote the manuscript. XL performed data analysis and wrote the manuscript. YZH, SW, SYW, JS, YCH, YL and SJX performed data analysis. JZ conceived the research and wrote the manuscript.

7 Acknowledgements

We would like to acknowledge the public databases including TCGA and GEO for their contributions to human medicine.

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Tables

Table 1

Table 2

Table 3