IL‐1RA suppresses esophageal cancer cell growth by blocking IL‐1α

Abstract Background Interleukin‐1 promotes tumor angiogenesis through VEGF production. The interleukin‐1 receptor antagonist can suppress tumors by blocking this effect. Methods Immunohistochemistry, WB, and gene sequencing were used to analyze the expression of IL‐1RA in esophageal cancer patients. WB was used to detect the expression of IL‐1RA and interleukin‐1α in esophageal cancer cells. Stable ESCC cell models overexpressing the IL‐1RA were constructed. Their cell functions were tested, and their effects on VEGF were examined. Results IL‐1RA is downregulated in primary EC tumors, and this downregulation of IL‐1RA is closely related to TNM staging and survival prognosis. The overexpression of IL‐1RA increased the proliferation of KYSE410 EC cells, which have a high level of IL‐1α expression. Overexpression of IL‐1RA in KYSE410 cells promotes a decrease in the expression of VEGF‐A. However, IL‐1RA expression did not cause any changes in EC9706 cells with low IL‐1α expression. Conclusion IL‐1RA acts as a tumor suppressor, and its deletion promotes tumor progression by increasing VEGF‐A expression in ESCC.


| INTRODUC TI ON
Esophageal cancer is the seventh most common form of malignant cancer worldwide and the sixth most common cause of cancer-related death. Notable forms of esophageal cancer include adenocarcinoma of the esophagus and esophageal squamous cell carcinoma (ESCC). 1,2 Although current treatments, such as surgery and chemotherapy, have progressed esophageal cancer treatment, the 5-year overall survival rate of ESCC is still low (20%-30%) due to metastasis, recurrence, and resistance to chemoradiotherapy. 3 Furthermore, the early symptoms and signs of esophageal cancer are difficult to detect; therefore, the disease is usually identified in the middle and late stages of pathogenesis. Subsequently, distant metastasis may occur, leading to poor prognosis. 4,5 Thus, exploring the mechanism of ESCC progression is critical to improve diagnosis, treatment, and prognosis.

IL-1RA suppresses esophageal cancer cell growth by blocking IL-1α
Sui Chen 1 | Zhimin Shen 1 | Zhun Liu 1 | Lei Gao 1 | Ziyang Han 1 | Shaobin Yu 1 | Mingqiang Kang 1,2,3 Sui Chen and Zhimin Shen contributed equally to this work. Interleukin-1 (IL-1), a pro-inflammatory chemokine, can promote the proliferation and differentiation of tumor cells via interactions with specific receptors on tumor cell membrane surfaces. 6 IL-1RA,   IL-1α, and IL-1β are all members of the interleukin-1 family. IL-1α   and IL-1β bind to the same receptor and perform similar biologi-cal activities, such as promoting tumor growth and metastasis via enhanced angiogenesis. This activity is performed by the regulation of the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF). 7   which was multiplied by the staining intensity (0, no staining; 1, weak staining, light yellow; 2, moderate staining, yellow-brown; and 3, strong staining, brown) to obtain a score ranging from 0 to 12. A score <3.5 was considered as low IL-1RA expression, while a score >3.5 was considered as high IL-1RA expression. All of the tissues were analyzed by two pathologists.

| Western blot analysis
Tissues or cells were lysed in Western & IP cell lysis buffer (Beyotime) with PMSF (Amresco) for 30 minutes on ice at 4°C followed by centrifugation at 12 000 × g for 15 minutes at 4°C.
The supernatants were collected, and the total protein concentration was measured using the BCA Protein Assay Kit (Thermo Scientific). Equimolar amounts of protein were loaded into each well and separated with 12% SDS-PAGE. Then, proteins were transferred to a 0.45-μm PVDF membrane (Amersham Hybond, GE Healthcare), which was blocked in 2% bovine serum albumin (Amresco) prior to overnight incubation overnight at 4°C with the following primary antibodies: rabbit anti-IL-1RA, rabbit anti-IL-1α ( method. The expression level was normalized against endogenous GAPDH. All the primers were designed by BioSune Biotechnology Co., Ltd. (Shanghai).

| Cell lines
Human EC cell lines KYSE410 and EC9706 were purchased from Hunan Fenghbio Biological Ltd, China. The cells were grown in RPMI-DMEM (Gibco) medium supplemented with 10% FBS (Gibco) and incubated at 37°C in an atmosphere of 5% CO 2 .

| Plasmids and generation of stable ec cell lines
The

| Wound-healing assay
Transfected cells were grown to 100% confluence in six-well plates.
The cell layers were scratched using a 20-μL tip to form wound gaps, washed three times with phosphate-buffered saline (PBS), and photographed at different time points. The cells were counted using a scale label to determine movement activity away from the original scratch location 48 hours after knockdown.

| Cell invasion assay
Cell invasion assays were performed using transwell membranes

| Colony formation assay
Stably transfected cells were harvested and seeded in six-well plates at a density of 1 × 10 3 cells/well.

| Statistical analysis
Statistical analysis was performed using SPSS 21.0 for Windows.
All data used for the analysis were expressed as the means ± SDs from three independent experiments. The association between IL-1RA expression and the clinicopathological parameters was analyzed using Pearson's chi-square test and Spearman's rank-order correlation. Survival curves were plotted using Kaplan-Meier analysis. Differences were considered significant when P-values were <0.05.

| Downregulation of IL-1RA in EC is correlated with poor prognosis
Seventy-six tumor samples were analyzed by immunohistochemistry (IHC) to detect the prognostic value of IL-1RA expression. As shown in representative Figure 1 and Table 1, IL-1RA expression was significantly lower in the EC tumors than in the adjacent normal tissues.
Western blot analysis and genetic testing also confirmed the expression of both IL-1RA protein ( Figure 1A) and mRNA ( Figure 1B (Table 1). After confirming that IL-1RA expression was downregulated in EC (P = 0.003), Kaplan-Meier analysis was used to investigate the relationship F I G U R E 3 A, Cell scratch test to detect cell migration ability. IL-1RA expression had no significant effect on cell migration ability. B, Cell plate clone assay to detect cell proliferation ability. IL-1RA overexpression significantly reduced the proliferation of KYSE410 cells, but no effect was observed for the EC9706 cells. C, CCK-8 assay to detect cell proliferation function. IL-1RA overexpression significantly reduced the proliferation of KYSE410 cells (P＜0.05), but no effect was observed for EC9706 cells between IL-1RA expression assessed by IHC and patient outcome.
As shown in Figure 1D, patients with low IL-1RA-expressing tumors had significantly shorter 5-year survival than patients with high IL-1RA-expressing tumors (P = 0.044). Altogether, these results suggest that IL-1RA may be a tumor suppressor and that a decrease in IL-1RA expression can promote the progression of EC.

| IL-1RA overexpression suppressed EC cell proliferation
To investigate the biological functions of IL-1RA in regulating ESCC, we first detected the expression of IL-1RA and IL-1α in EC cell lines ( Figure 2A). Then, we used gene overexpression strategies to specifically increase the amount of IL-1RA protein in the EC cell lines KYSE410 and EC9706. Stable overexpression of IL-1RA in these cells was confirmed by Western blot analysis ( Figure 2B). The overexpression of IL-1RA did not produce any changes in the rate of migration of KYSE410 and EC9706 cells in vitro, as evaluated by wound-healing assay ( Figure 2C). Then, we compared the effect of IL-1RA on cell proliferation using CCK-8 and colony formation assays. As shown in Figure 2D and Figure 2E, proliferation was suppressed by overexpression of IL-1RA in KYSE410 cells, which express IL-1α. No difference was observed in EC9706 cells.

| IL-1RA suppressed ESCC proliferation via the IL-1α/VEGF signaling pathway
To understand the molecular mechanisms underlying IL-1RA-in-

| D ISCUSS I ON
The results reported in this study provide evidence that the ex- The development of new blood vessels is a key pathway in the progression of solid tumors. Angiogenesis not only provides the tumor with oxygen and essential nutrients but also promotes the spread and proliferation of cancer cells. 14 Angiogenesis is a complex, multi-step process involving extracellular matrix remodeling, migration and proliferation of endothelial cells, and capillary formation. 15 VEGF is the prototypical angiogenic stimulating molecule that has been implicated in several steps of the angiogenic process. Increasing VEGF expression is directly proportional to increasing tumor growth and metastasis by promoting angiogenesis and increasing vascular permeability. 16 The role of VEGF in angiogenesis was further confirmed by the effects of hypoxia and several indirect effects of proangiogenic factors, which were able to further increase the synthesis of VEGF. 17,18 It is important that VEGF has also been associated with tumor progression by stimulating angiogenesis in human esophageal carcinoma. 19,20 IL-1RA is a member of the interleukin-1 family. In contrast to IL-1α and IL-1β, which promote tumor progression through inflammation, IL-1RA possesses a tumor-suppressing effect by blocking the binding of IL-1 to its target receptor [11,12]. Increasing in the EC9706 cells, which naturally have low IL-1α expression. All our experiments indicate that IL-1RA may inhibit the angiogenesis of EC cells by inhibiting the IL-1α/VEGF signaling pathway. In addition, anakinra (Kineret), an IL-1 receptor antagonist that blocks the biologic activity of IL-1, has been confirmed to be safe to inject into the human body. 25,26 Thus, anakinra has been approved as a clinical drug for the treatment of symptoms of rheumatoid arthritis and hidradenitis suppurative. 27,28 In summary, our study found that IL-1RA acted as a tumor suppressor in EC via regulation of the IL-1α/VEGF signaling pathway; therefore, IL-1RA might serve as a potential molecular target for EC treatments.