白细胞介素-1受体Ⅱ在结肠癌中的表达特征及其对肿瘤微环境的影响

Expression characteristics of interleukin-I receptor 2 in colon cancer and its impact on tumor microenvironment

目的观察人白细胞介素-1受体Ⅱ(IL-1R2)在肠癌肿瘤微环境(TME)中的表达特征,探究IL-1R2在结肠癌中的作用。方法分析基因表达谱数据库(GEO)中的结肠癌单细胞转录组测序(scRNA-seq)数据,确定IL-1R2基因在肿瘤组织中的表达分布。采用随机数字表法将小鼠分为对照组和IL-1R2敲基因组(Il1r2^(-/-))。利用氧化偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)制备结肠炎相关肠癌模型。在AOM/DSS诱导后的第70d,收集肿瘤组织,计数肿瘤数量并测量其直径;应用流式细胞术分析Il1r2^(-/-)小鼠和对照组小鼠TME中各类细胞群体浸润的差异以及免疫检查点分子的表达差异。采用独立样本t检验比较两组间差异。使用双向方差分析来比较体重曲线。结果小鼠结肠癌和人类结肠癌CD45^(+)免疫细胞的scRNA-seq数据的分析结果显示,与正常组织比较,IL-1R2在结肠癌组织中高表达。Il1r2^(-/-)小鼠与对照组小鼠肿瘤数量之间的差异无统计学意义(4.750±0.977比7.800±1.114,t=2.005,P>0.05),但是Il1r2^(-/-)小鼠肿瘤负荷显著低于对照组(24.170±7.483比96.000±19.990,t=3.618,P<0.01)。Il1r2^(-/-)小鼠TME中CD45^(+)免疫细胞(35.140±3.673比7.076±1.929,t=6.764,P<0.001)和CD8^(+)T细胞(26.820±1.004比15.900±2.665,t=3.835,P<0.05)的比例显著高于对照组,CD4^(+)T细胞的比例有下降趋势,差异无统计学意义(P>0.05)。IL-1R2缺失导致程序性死亡因子-1(PD-1)^(+)CD4^(+)T细胞(14.340±3.401比24.570±2.330,t=2.482,P<0.05)、T细胞免疫球蛋白黏蛋白-3(Tim-3)+CD4^(+)T细胞(20.740±4.001比41.500±4.108,t=3.620,P<0.01)、PD-1^(+)Tim-3^(+)CD4^(+)T细胞(7.184±2.129比15.140±1.730,t=2.900,P<0.05)比例显著低于对照组,而CD8^(+)T细胞上PD-1和Tim-3的表达差异无统计学意义(P>0.05)。结论IL-1R2在结肠癌中高表达,IL-1R2缺失可以降低小鼠对结肠炎相关肠癌的易感性。

Objective To elucidate the expression characteristics of interleukin-1 receptor 2(IL-1R2)in the tumor microenvironment(TME)of colon cancer and explore the role of IL-1R2 in colon cancer.Methods The single-cell RNA sequencing(scRNA-seq)data of colon cancer in the gene expres-sion omnibus(GEO)database were analyzed to determine the expression distribution of IL-1R2 gene in tumor tissues.Mice were randomly divided into control group and IL-1R2 knockout(Il1r2^(-/-))group.Azoxymethane(AOM)and dextran sodium sulfate(DSS)were used to induce colitis-associated colon canc-er models.On day 70 after AOM/DSS,tumor tissues were collected,and the number and diameter of tumors were counted and measured.Flow cytometry was used to analyze the differences in the infiltration of various cell populations and expression of immune checkpoint molecules in the TME between Il1r2^(-/-)mice and control mice.Independent sample t-test was used to compare the differences between the two groups.Two-way ANOVA was used to compare weight curves.Results The scRNA-seq data of CD45^(+)immune cells from mouse colon cancer and human colon cancer showed that IL-1R2 was highly expressed in colon cancer tissues as compared with normal tissues.There was no significant difference in the number of tumors between Il1r2^(-/-)mice and control mice(4.750±0.977 vs.7.800±1.114,t=2.005,P>0.05),but the tumor load of Il1r2^(-/-)mice was significantly lower than that of control group(24.170±7.483 vs.96.000±19.990,t=3.618,P<0.01).As compared with control mice,Il1r2^(-/-)significantly increased CD45^(+)immune cells in TME(35.140±3.673 vs.7.076±1.929,t=6.764,P<0.001)and CD8^(+)T cells(26.820±1.004 vs.15.900±2.665,t=3.835,P<0.05),the proportion of CD4^(+)T cells showed a decreasing trend,and the difference was not statistically significant(P>0.05).Compared with the control group,IL-1R2 deletion resulted in programmed death 1(PD-1)^(+)CD4^(+)T cells(14.340±3.401 vs.24.570±2.330,t=2.482,P<0.05),T cell immunoglobulin domain and mucin domain-containing molecule-3(Tim-3)^(+)CD4^(+)T cells(20.740±4.001 vs.41.500±4.108,t=3.620,P<0.01),PD-1^(+)Tim-3^(+)CD4^(+)T cells(7.184±2.129 vs.15.140±1.730,t=2.900,P<0.05),while the expression of PD-1 and Tim-3 on CD8^(+)T cells was not significantly different(P>0.05).Conclusion IL-1R2 is highly expressed in colon cancer,and IL-1R2 deficiency can reduce the susceptibility to colitis-associated colon cancer in mice.