{"paper_id":"a70d65b7-fb47-43d5-99ff-988596866ff3","body_text":"Gastric cancer (GC) is the fifth most commonly diagnosed cancer and the third leading\ncause of cancer-associated mortality in men and women worldwide [ 1 ]. Patients with GC exhibit high metastasis and\nmortality rates and low early diagnosis rates [ 2 ]. Most patients with GC have advanced-stage disease at diagnosis and\nhave missed the optimum surgical window. Although there are many treatment methods\nfor GC, including surgery, chemotherapy, radiotherapy, and immunotherapy, the 5-year\nsurvival rate of patients remains poor [ 3 ].\nTherefore, it is important to identify new targets for the diagnosis, treatment, and\nprognosis of GC.\nThe interleukin-1 receptor type II ( IL-1R2 ) gene is located on the\nlong arm of chromosome 2 at band 2q12 in humans. The IL-1R2 protein is the\npredominant IL-1 receptor, and is 398 amino acids in length [ 4 ]. However, IL-1R2 differs from all other members of the IL-1\nreceptor family because it lacks a TIR domain and only has a short cytoplasmic tail\nof only 29 amino acids in length. As a decoy receptor, IL-1R2 cannot signal by\ncompetitive binding to IL-1β and preventing its binding to IL-1R1 [ 5 , 6 ].\nIL-1R2 is mainly expressed in neutrophils, B cells, monocytes, and macrophages.\nPlasma levels of soluble IL-1R2 are between 5 and 10 ng/ml in healthy donors and\nincrease in patients with infectious conditions [ 7 ]. IL-1R2 plays a role in a variety of diseases, including chronic skin\ninflammation, arthritis, endometriosis, and heart transplantation or autoimmune\nmyocarditis [ 7 ]. Moreover, IL-1R2\noverexpression is observed in a variety of tumors, and is indicative of poor\nprognosis, in breast cancer [ 8 ], colorectal\ncancer [ 9 ], pancreatic cancer [ 10 ], lung cancer [ 11 ], and oral cancer [ 12 ].\nHere, we investigated the clinical significance of IL-1R2 expression in human GC\ntissues and plasma, the relationship between IL-1R2 expression and\nclinicopathological factors, and evaluated its prognostic value.\n\nThe GC tissue array (Catalog number: HStmA180Su15, 98 cancer tissues, 82 normal\ntissues) was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China).\nThe array contained samples from 98 GC patients that underwent surgery between\nJuly 2006 and April 2007. The age of the patients ranged from 41 to 81 years,\nwith a median age of 65 years. Patients were followed up for 8.2–9.0\nyears. No patient received preoperative radiotherapy or chemotherapy. All\npatient surgical specimens were confirmed as GC by pathologists using\nHematoxylin and Eosin (H&E) staining. After excluding the incomplete\ntissue points and several missing tissue points when performing heated antigen\nretrieval, 88 cancer cases and 75 normal tissues were analyzed. The correlation\nbetween GC tissue IL-1R2 expression levels and the patients’ clinical\nparameters are listed in  Table 1 .\nThe goat polyclonal antibody against human IL-1R2 (AF263, diluted in 1:1) was\npurchased from R&D Systems (Minneapolis, MN, U.S.A.). HRP-conjugated\nanti-goat IgG polymer (PV-9003) was obtained from ZSGB-BIO (Beijing, China). The\nHuman IL-1R2 Quantikine ELISA Kit (DR1B00) was purchased from R&D Systems\n(Minneapolis, MN, U.S.A.). The RNeasy Mini Kit was supplied by Qiagen (Valencia,\nCA, U.S.A.), and SYBR Green Master Mix kits were provided by TaKaRa (Dalian,\nChina). DMEM and fetal bovine serum (FBS) were purchased from Gibco (Cambrex,\nMD, U.S.A.).\nImmunohistochemistry assays were used to detect IL-1R2 protein expression levels\nin human GC and normal gastric tissues. The paraffin-embedded tumor tissue\nmicroarray block was sectioned into 3 μm sections, dewaxed with xylene,\nand rehydrated with a series of graded alcohols. Tissue sections were heated at\n100°C for 30 min in EDTA (1 mM, pH 9.0) for antigen retrieval. After\ncooling, the tissue sections were immersed in 0.3% hydrogen peroxide\nsolution for 15 min to block endogenous peroxidase activity, washed with PBS for\n5 min, and blocked with 3% BSA solution at room temperature for 20 min.\nThe goat anti human IL-1R2 polyclonal antibody was incubated overnight at\n4°C, and then incubated with the HRP-conjugated anti-goat IgG polymer.\nDiaminobenzene was used as the chromogen and Hematoxylin was used as the nuclear\ncounterstain. Finally, the sections were dehydrated, cleared, and mounted.\nTwo pathologists with no knowledge of the patients’ information\nindependently examined the stained sections. IL-1R2 staining in the GC tissue\narray was assessed according to the H-score method as described in our previous\nreport [ 13 ]. The results were calculated\nas: H-score = %tumor cells unstained × 0 + %tumor\ncells stained weak × 1 + %tumor cells stained moderate × 2\n+ %tumor cells stained strong × 3. H-scores ranged from 0\n(100% negative tumor cells) to 300 (100% strongly stained tumor\ncells). Results from the two pathologists were averaged and used for statistical\nanalysis.\nThe enzyme-linked immunosorbent assay (ELISA) was performed to detect IL-1R2\nlevels in GC patients and healthy people. Twenty-eight serum samples from\nhealthy people and 50 serum samples from patients with GC were obtained from the\nThird Affiliated Hospital of Soochow University. Informed consent was obtained\nfrom all patients, and the study was approved by the institution’s ethics\ncommittee. All assays were performed according to the manufacturer’s\ninstructions.\nGES1 and human GC cell lines (AGS, MGC803, BGC823, and SGC7901) were obtained\nfrom the Chinese Academy of Sciences, Shanghai Institutes for Biological\nSciences, and were cultured in standard DMEM supplemented with 10% FBS\nand antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin)\nunder standard culture conditions (5% CO 2 , 37°C).\nReal-time PCR (RT-PCR) was performed to evaluate  IL-1R2  mRNA\nexpression in GC tissues and cells. GC tissue and para-cancer tissue was\nobtained from the Gastrointestinal Surgery Department of the Third Affiliated\nHospital of Soochow University. Informed consent was obtained from all patients,\nand the study was approved by the institution’s ethics committee. Total\nRNA was extracted from GC tissues and cells using TRIzol reagent (Invitrogen)\nand reverse transcribed into cDNA using an RT reaction kit (Promega). RT-PCR was\nperformed to detect relative RNA expression using the SYBR green method and an\nABI 7500 real-time PCR system (Applied Biosystems, U.S.A.). Human GAPDH was\nselected as an internal reference gene. GAPDH and IL-1R2 primer sequences used\nwere: GAPDH forward primer, 5′-GGAGCGAGATCCCTCCAAAAT-3′; GAPDH\nreverse primer, 5′-GGCTGTTGTCATACTTCTCATGG-3′; IL-1R2 forward\nprimer, 5′-GCCAATGACACCCACATAGAGAGC-3′, and IL-1R2 reverse primer,\n5′-GGAAGAGCGAAACCCACAGAGTTC-3′. The relative IL-1R2 mRNA\nexpression was calculated using the\n2 −ΔΔ C T \nmethod.\nAll data were analyzed using the GraphPad Prism 8.0 software package (GraphPad\nSoftware, Inc., San Diego, U.S.A.) and R 3.6.3. IL-1R2 protein expression in GC\nand normal tissue, IL-1R2 mRNA expression in GC and para-cancer tissue, and\nserum IL-1R2 levels in GC and healthy controls were tested using the unpaired\nStudent’s  t  test. The association between IL-1R2\nexpression and the clinical pathological characteristics was assessed using the\nchi-square test. R package survival was used for single-factor and multi-factor\nanalyses. Kaplan–Meier and log-rank tests were used to compare the\noverall survival rate of patients with different clinicopathological parameters.\nCox proportional hazards model was used to estimate the risk (HR) among\ndifferent clinicopathological parameters, IL-1R2 expression and death risk with\n95% confidence interval (CI). A  P -value <0.05 was\nconsidered statistically significant.\n\nImmunohistochemical staining was used to detect IL-1R2 expression in GC tissues\nand normal gastric tissues. IL-1R2 expression was higher in GC tissues than in\nnormal gastric tissue, and IL-1R2 positive staining was mainly located on the GC\ncell cytoplasm ( Figure 1 A). No, or weak,\nstaining was observed in normal gastric tissues ( Figure 1 B). The H-score of IL-1R2 staining in GC tissues was\nsignificantly higher than that in normal tissues\n( P =0.011,  Figure\n2 A). However, there was no correlation between IL-1R2 expression and any\nclinical parameter in GC patients ( Table\n1 ).\n( A ) High IL-1R2 expression in human GC tissues, and positive\nstaining of IL-1R2 might be found in the GC cell cytoplasm.\n( B ) The IL-1R2 was not expressed in normal gastric\ntissues.\n( A ) H-score of IL-1R2 staining in GC tissues was\nsignificantly higher than that in normal gastric tissues.\n( B ) IL-1R2 expression was not associated with overall\nsurvival in GC patients. ( C ) IL-1R2 expression was not\nassociated with overall survival in GC patients of TNM stages I and II.\n( D ) Overall survival in TNM stages III and IV patients\nwith high IL-1R2 expression was significantly poorer than that in\npatients with low IL-1R2 expression\n( P =0.03).\nTo study the relationship between IL-1R2 expression and prognosis in GC, we\nperformed Kaplan–Meier survival analysis. The results showed that IL-1R2\nexpression was not associated with overall survival in GC patients\n( P =0.55,  Figure\n2 A). Overall survival in TNM stages III and IV patients with high\nIL-1R2 expression was significantly poorer than that in patients with low IL-1R2\nexpression ( P =0.03,  Figure\n2 D). Multi-factor Cox analysis indicated that increased age (HR\n= 1.0324, 95% CI: 1.0031–1.062,\n P =0.030), TNM stage III + IV (HR = 4.489,\n95% CI: 1.8343–10.986,  P =0.001), and high\nIL-1R2 expression (HR = 2.151, 95% CI: 1.198–3.861,\n P =0.010) were independent prognostic factors of GC\n( Table 2 ).\nBold signifies  P <0.05.\nTo detect the serum IL-1R2 levels of GC patients, we analyzed the levels of serum\nsoluble IL-1R2 in GC patients ( n =50) and healthy\ncontrols ( n =28). Using ELISA, we found that the serum\nIL-1R2 levels in GC patients were significantly higher than that in healthy\ncontrols ( P <0.05,  Figure\n3 ).\nELISA analysis indicated that the serum level of IL-1R2 in GC patients\nwas significantly higher than that in healthy controls.\nIL-1R2  mRNA expression in the GC and para-cancer tissues of nine\npatients with GC was measured by RT-PCR.  IL-1R2  mRNA levels\nwere higher in GC tissues than in para-cancer tissues\n( P =0.0191,  Figure\n4 A). We detected  IL-1R2  mRNA levels in GC cells. RT-PCR\nrevealed that the IL-1R2 mRNA levels in most GC cells (AGC, MGC803, and BGC823;\n P <0.01,  P <0.05, and\n P <0.05, respectively) were higher than that in GES1,\nwith the exception of SGC7901 ( P <0.05).\n( A )  IL-1R2  mRNA levels were higher in GC\ntissues than in para-cancer tissues ( P =0.0191).\n( B ) The IL-1R2 mRNA levels in AGC, MGC803, and BGC823\ncells was higher than that in GES1 ( P <0.01,\n P <0.05 and  P <0.05,\nrespectively), however, the IL-1R2 mRNA level in SGC7901 cell was lower\nthan that in GES1 ( P<0.05 ).\n* P <0.05,\n** P <0.01.\n\nIL-1 is the key mediator of innate and adaptive immunity. IL-1 plays a key role in\npromoting inflammatory response by shaping different components of the tumor\nmicroenvironment, including tumor infiltration, myeloid cell recruitment,\nangiogenesis, and inhibition of anti-tumor immunity [ 14 ]. IL-1 plays a major role in the development of several autoimmune\ndiseases and cancers, including type 2 diabetes [ 15 ], Alzheimer’s disease [ 16 ], esophageal cancer [ 17 ], and\nbreast cancer [ 8 ]. Therefore, inhibition of\nIL-1 signal transduction is an important therapeutic approach for both cancer and\nautoimmune diseases [ 18 ], and IL-1R2 can\ninhibit IL-1 signal transduction. In the IL-1 negative regulatory system, IL-1R2\nacts as a decoy receptor and a negative regulator, suggesting that it plays a\ncrucial role in the IL-1-mediated immune response [ 19 ]. IL-1R2 exists in two isoforms, membrane-bound protein and soluble\nprotein [ 20 ]. Plasma soluble IL-1R2 levels in\nhealthy blood donors are between 5 and 10 ng/ml, and plasma soluble IL-1R2 levels\nare elevated in infected patients [ 21 ].\nIL-1R2 is expressed in a variety of diseases and tumors, and plays a role in\npromoting cancer in most tumors. The expression of intracellular IL-1R2 in human\ncolorectal cancer cells is higher than that in normal colon cells. Intracellular\nIL-1R2 regulates IL-6 and VEGF-A expression and the migration and proliferation of\ncolorectal cancer cells [ 9 ]. Zhang et al.\nshowed that IL-1R2 is up-regulated in breast cancer tissues, and IL-1R2 increases\nBMI1 deubiquitination and stability by binding to and enhancing the activity of\nubiquitin-specific protease 15, promoting breast cancer cell proliferation and\ninvasion. Meanwhile, IL-1R2‐neutralizing antibody significantly inhibits\ncancer cell growth, invasion, and chemoresistance  in vitro  [ 8 ]. Moreover, the level of IL-1R2 in\ninfiltrating Treg cells is higher in colorectal cancer tissues than in normal\ntissues. IL-1R2 up-regulation may be another mechanism through which tumor-resident\nTreg cells inhibit anti-tumor immune response through neutralizing effector cell\nIL-1β function [ 22 ]. These studies\nsuggest that IL-1R2 plays an important role in regulating the biological behavior of\ncancer cells. Therefore, IL-1R2 is a potential clinical biomarker for human cancer,\nand a potential new therapeutic target for the treatment of cancer.\nIn the present study, IL-1R2 expression in GC and normal gastric tissues was detected\nusing tissue microarray and immunohistochemistry. Our results show that IL-1R2\nexpression in GC tissues is higher than that in normal gastric tissues, and that\nthere was no significant correlation between IL-1R2 expression and the\nclinicopathological characteristics of GC. Survival analysis revealed that IL-1R2\nexpression was not associated with overall survival in patients with GC. However, in\npatients with TNM stages III and IV GC, overall survival was significantly poorer in\nthose with high IL-1R2. We investigated the expression of IL-1R2 in GC tissues,\ncells, and serum using RT-PCR and ELISA assays. We found that IL-1R2 mRNA levels was\nhigher in GC tissues than in para-cancer tissues, and that the level of IL-1R2 mRNA\nin most GC cells was higher than that in GES1 control cells. The plasma soluble\nIL-1R2 levels were higher in the GC group than in the healthy control group.\nCollectively, our results show that increased IL-1R2 is involved in the progression\nof human GC. The potential contribution of IL-1R2 examination in immunotherapeutic\napproaches against human GC and the underlying mechanisms of IL-1R2 in GC\nprogression need to be further investigated.\n\nOur results suggest that increased IL-1R2 is involved in the initiation and\nprogression of human GC, suggesting that IL-1R2 could be a potential predictor of\ntherapy against human GC.","source_license":"CC-BY-4.0","license_restricted":false}