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结直肠癌是危害人类健康的第三大恶性肿瘤,其高发病率(每年全球新发病例1 096 601例,占所有癌症的6.1%)和高死亡率(每年全球死亡病例551 269例,占所有癌症的5.8%),是全球公共卫生面临的重大挑战[1]。近年来,对结直肠癌分子机制研究取得了重大的进展,但是由于结直肠癌的高转移率及高复发率,结直肠癌仍然是癌症主要的死因;基于此,亟需找到一种可用于结直肠癌治疗预后的预测工具[2]。先前的大多数相关研究都是基于临床病理特征(如肿瘤大小、肿瘤数量、淋巴结及血管浸润等)和单分子生物标志物[如癌胚抗原(CEA)、糖类抗原(CA199)、CH24和CA242等]构建的预后模型[3-4];尽管如此,仍没有找到一种可靠的指标可准确用于结直肠癌治疗预后的预测。
随着对结直肠癌发生、发展机制研究的深入,发现免疫反应在结直肠癌的恶性进展中起着重要的作用。免疫反应对癌症的产生具有双向作用,正常条件下癌组织作为一种异常的器官可被免疫系统消灭清除;但在某些情况下却可以促进癌变[5-6]。结直肠癌中存在着大量的异质性,在克隆选择的作用下,基因组不稳定性使结直肠癌产生不同的细胞群体[7]。新的细胞群体间具有不同的免疫特性,突变的肿瘤细胞通过躲避免疫系统的攻击获得无限增殖的能力,使肿瘤细胞异质化的分子事件可能促进癌症的发生和发展[8-9]。
近年来,相关研究发现,在乳腺癌、前列腺癌及肝癌等肿瘤中联合多个基因所作的预后模型可显著提高预后预测的准确性;免疫相关基因在结直肠的恶性进展中具有重要作用,然而关于免疫基因所作的预后模型在结直肠癌中尚缺乏相关的研究[10-12]。本研究利用预后相关免疫基因建立预后模型,首次研究了多种免疫基因联合在结直肠癌预后预测中的优势;并分析了结直肠癌中免疫细胞及转录因子及免疫基因的相互作用关系。本研究摒弃了单基因预后预测的敏感性低或灵敏度低的缺陷,有望为结直肠癌的治疗及预后提供一个可靠的指标,并为结直肠癌的免疫反应研究提供了一定的参考。
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以|LogFC|>1, P < 0.05的条件筛选,结果发现有6 478个基因在结直肠癌中差异表达(其中1 716个基因表达下调,4 762个基因表达上调)。引用R软件“pheatmap”包对差异基因绘制热图(见图 1A)及火山图(见图 1B)。
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从ImmPort下载结直肠癌相关免疫基因(共包含2 498个免疫相关基因),安装Perl软件对免疫基因与结直肠癌中的差异表达基因进行取交集,筛选出在结直肠癌中差异表达的免疫基因(共包含467个差异表达的免疫基因),引用R软件“pheatmap”包对差异表达的免疫基因绘制热图(见图 2A)、火山图(见图 2B)。
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从TCGA数据库下载结直肠癌的临床病理数据,然后将病人生存时间与结直肠癌中差异表达免疫基因进行合并。引用R软件“survival”包,通过单因素COX分析筛选出预后相关的免疫基因(包含50个预后相关免疫基因,其中11个低风险比基因,39个高风险比基因),对结果绘制森林图(见图 3)。风险比(hazard ratio,HR)[HR=暴露组的风险函数h1(t)/非暴露组的风险函数h2(t), t指在相同的时间点上]。
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通过TF网站,下载肿瘤相关转录因子(共包含318个肿瘤相关转录因子)。将肿瘤相关转录因子与结直肠癌差异表达基因取交集,获得在结直肠癌中差异表达的肿瘤相关转录因子(共有68个差异转录因子,其中23个转录因子表达下调,45个转录因子表达上调)。并引用R软件“pheatmap”包绘制热图(见图 4A)、火山图(见图 4B)。
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引用R软件“survival”包对结直肠癌中差异免疫基因作多因素COX分析,筛选出可作为结直肠癌独立预后风险因子的差异免疫基因(共包含18个差异免疫基因,其中4个基因与预后呈负相关,14个基因与预后呈正相关)及个模型基因的风险系数(risk coefficient)(见表 1)。构建免疫基因模型风险评分=(CoefficientmRNA1×mRNA1的表达)+(CoefficientmRNA2×mRNA2的表达)+......+(CoefficientmRNAn×表达mRNAn)。
ID coef HR HR.95L HR.95H P CD1B -2.155 976 693 0.115 790 044 0.022 328 11 0.600 468 834 0.010 248 76 SLC10A2 0.782 264 364 2.186 417 51 1.351 351 517 3.537 511 497 0.001 440 093 FABP4 0.008 285 932 1.008 320 355 1.000 117 572 1.016 590 416 0.046 792 537 FGF2 0.337 506 581 1.401 448 831 1.144 974 262 1.715 373 778 0.001 065 103 CCL28 -0.095 694 683 0.908 741 428 0.856 395 586 0.964 286 828 0.001 570 311 IGHG1 -0.000 732 798 0.999 267 471 0.998 592 4 0.999 942 998 0.033 562 553 IGHV4-31 0.013 604 819 1.013 697 786 1.005 661 675 1.021 798 113 0.000 807 408 IGKV1-6 0.008 862 377 1.008 901 764 1.004 108 388 1.013 718 023 0.000 265 007 IGKV1-8 0.042 254 831 1.043 160 274 1.006 934 751 1.080 689 048 0.019 119 346 ESM1 0.214 226 484 1.238 903 215 1.144 090 679 1.341 573 01 0.000 000 134 STC2 0.043 707 547 1.044 676 792 0.997 685 046 1.093 881 885 0.062 706 293 TNFSF12 0.071 779 075 1.074 417 952 0.983 332 525 1.173 940 56 0.112 265 354 UCN 0.436 580 71 1.547 407 129 1.229 882 742 1.946 908 222 0.000 1946 69 UTS2 0.165 410 499 1.179 877 358 0.965 107 433 1.442 441 051 0.106 631 656 VIP 0.084 268 737 1.087 921 219 1.044 614 328 1.133 023 497 0.000 047 8 GLP2R -4.612 686 23 0.009 925 121 0.000 631 273 0.156 046 663 0.001 032 719 IL1RL2 0.181 334 897 1.198 816 592 1.038 151 643 1.384 346 141 0.013 512 859 TRDC 0.111 556 883 1.118 017 339 1.009 457 521 1.238 251 976 0.032 307 538 注:风险系数coef表示基因对模型风险评分的贡献,负值表示基因与预后呈负相关,正值表示基因与预后呈正相关 表 1 结直肠癌中预后模型免疫基因的筛选
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风险评分生存分析见图 5A,ROC曲线见图 5B。引用“pheatmap”包分析每例病人的风险评分与其生存状态的关系,评价预后模型对评估病人预后的价值;并绘制生存状态图(见图 5C)、风险曲线(见图 5D)和风险热图(见图 5E)。结果显示,高风险组相较于低风险组预后较差(P < 0.05);预后模型对评估病人预后性能较好(AUC=0.861);风险评分高的病人总体预后较差,该模型对病人预后显示出较为可靠的价值。
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将预后模型风险评分及结直肠癌病人临床病理特征进行合并,引用R软件“survival”包对预后模型及病人临床病理特征作单因素(见图 6A)及多因素(见图 6B)COX分析,分析预后模型及临床病理特征对结直肠癌病人的独立预后作用,并绘制森林图对结果进行可视化。结果显示,免疫基因预后模型、TNM分期及病人及年龄可做为评估病人预后的独立风险因子。
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为进一步研究结直肠癌中转录因子和免疫基因的相关性,引用R软件对结直肠癌中的差异转录因子及差异免疫基因作相关性检验。按相关系数|R|>0.4, P < 0.001的条件对结果进行筛选,并应用cytoscape软件作转录因子和免疫基因的调控网络图(见图 7)进行可视化(线条多少表示相关性强弱,线条越多相关性越强,线条越少相关性越差)。
结果显示,SLIT2、INHBA、SEMA3G、PLCG2等免疫基因与转录因子相关性较强;CCL28、CD1B等免疫基因与转录因子相关性较差。LMC2、IKZF1、IRF4等转录因子与免疫基因相关性较强;KLF4、CDK2、EZH2等转录因子与免疫基因相关性较差。由此,我们推测转录因子通过与免疫基因相互作用在结直肠癌中发挥作用。
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通过TIMER下载TCGA数据库中的各结直肠癌病例中免疫细胞的含量;并利用R软件对免疫细胞的含量与预后模型风险进行相关性分析,结果以散点图进行可视化。结果显示,风险评分与B细胞(见图 8A)无明显相关性,与CD4-T细胞(见图 8B)、CD8-T细胞(见图 8C)、树突状细胞(见图 8D)、巨噬细胞(见图 8E)及中性粒细胞(见图 8F)呈正相关,|R|>0.1, P < 0.05。结果表明,免疫基因可影响免疫细胞的产生,并协同促进结直肠癌的发生发展。
多免疫基因预后模型在评估结直肠癌生存和预后中的作用:基于TCGA数据库的研究
Role of multi-immune gene prognosis model in evaluating the survival and prognosis of colorectal cancer: a study based on the TCGA database
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摘要:
目的通过生物信息学方法筛选与结直肠癌预后相关的免疫基因并构建多免疫基因预后模型, 以期为结直肠癌生存和预后的评估提供重要的临床资料, 并为肿瘤免疫在结直肠癌中的研究提供思路。 方法从TCGA数据库下载结直肠癌基因表达数据和临床病理数据, 对其进行差异分析后获得差异表达的基因; 从ImmPort免疫基因数据库下载免疫基因, 与结直肠癌差异基因取交集后获得差异表达的免疫基因; 进一步对差异免疫基因进行生存分析获得结直肠癌预后相关基因, 以此构建多免疫基因预后模型(预后模型); 然后, 通过生存分析预后模型风险评分对结直肠癌生存的影响, 利用ROC分析以及绘制风险曲线验证预后模型风险评分在评估结直肠癌预后中的准确性; 通过独立预后分析预后模型风险评分是否可作为评估病人预后的独立风险因子; 最后, 分析免疫基因与转录因子及免疫细胞的相关性。 结果免疫基因预后模型风险评分高风险组预后较差(P < 0.01);预后模型风险评分能对结直肠病人预后进行准确分组, 并且在对结直肠预后的分析中具有较高的准确性(AUC=0.861);免疫基因与转录因子以及免疫细胞之间存在一定的相关性。 结论预后模型能准确评估病人预后, 并且高风险组病人的生存期显著低于低风险组; 免疫基因可能通过调节转录因子以及免疫细胞的产生进而调节肿瘤的恶性进展, 该研究阐明了免疫因素对结直肠癌预后的影响, 为结直肠癌在免疫方向的研究提供了参考。 Abstract:ObjectiveTo screen the immune genes related to the prognosis of colorectal cancer through bioinformatics and construct a multi-immune gene prognosis model, so as to provide important clinical data for the evaluation of survival and prognosis of colorectal cancer, and to provide ideas for the study of tumor immunity in colorectal cancer. MethodsThe gene expression data and clinicopathological data of colorectal cancer were downloaded from TCGA database, and the differentially expressed genes were obtained after differential analysis.The immune genes were downloaded from ImmPort immune gene database, and the differentially expressed immune genes were obtained after intersection with differential genes of colorectal cancer.Further, survival analysis of differential immune genes was carried out to obtain prognosis-related genes of colorectal cancer, so as to build a multi-immune gene prognosis model (prognosis model).Then, the effect of prognosis model risk score on the survival of colorectal cancer was evaluated by survival analysis, ROC analysis and drawing the risk curve were used to verify the accuracy of the prognosis model risk score in assessing the prognosis of colorectal cancer, and whether the prognostic model risk score being an independent risk factor for the prognosis of patients was evaluated through independent prognostic analysis.Finally, the correlation between immune genes, transcription factors and immune cells was analyzed. ResultsThe prognosis of the high-risk group with immune gene prognosis model risk score was poor (P < 0.01).The prognostic model risk score could accurately group the prognosis of colorectal patients, and had high accuracy in the analysis of colorectal prognosis (AUC=0.861).There is a certain correlation between immune genes, transcription factors and immune cells. ConclusionsThe prognosis model can accurately assess the prognosis of patients, and the survival time of patients in the high-risk group is significantly lower than that of the low-risk group.Immune genes may regulate the malignant progression of tumors by regulating the production of transcription factors and immune cells.This study clarifies the influence of immune factors on the prognosis of colorectal cancer, and provides the reference for the research of colorectal cancer in the field of immunity. -
Key words:
- colorectal neoplasms /
- prognosis model /
- immune genes /
- transcription factors /
- immune cells
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表 1 结直肠癌中预后模型免疫基因的筛选
ID coef HR HR.95L HR.95H P CD1B -2.155 976 693 0.115 790 044 0.022 328 11 0.600 468 834 0.010 248 76 SLC10A2 0.782 264 364 2.186 417 51 1.351 351 517 3.537 511 497 0.001 440 093 FABP4 0.008 285 932 1.008 320 355 1.000 117 572 1.016 590 416 0.046 792 537 FGF2 0.337 506 581 1.401 448 831 1.144 974 262 1.715 373 778 0.001 065 103 CCL28 -0.095 694 683 0.908 741 428 0.856 395 586 0.964 286 828 0.001 570 311 IGHG1 -0.000 732 798 0.999 267 471 0.998 592 4 0.999 942 998 0.033 562 553 IGHV4-31 0.013 604 819 1.013 697 786 1.005 661 675 1.021 798 113 0.000 807 408 IGKV1-6 0.008 862 377 1.008 901 764 1.004 108 388 1.013 718 023 0.000 265 007 IGKV1-8 0.042 254 831 1.043 160 274 1.006 934 751 1.080 689 048 0.019 119 346 ESM1 0.214 226 484 1.238 903 215 1.144 090 679 1.341 573 01 0.000 000 134 STC2 0.043 707 547 1.044 676 792 0.997 685 046 1.093 881 885 0.062 706 293 TNFSF12 0.071 779 075 1.074 417 952 0.983 332 525 1.173 940 56 0.112 265 354 UCN 0.436 580 71 1.547 407 129 1.229 882 742 1.946 908 222 0.000 1946 69 UTS2 0.165 410 499 1.179 877 358 0.965 107 433 1.442 441 051 0.106 631 656 VIP 0.084 268 737 1.087 921 219 1.044 614 328 1.133 023 497 0.000 047 8 GLP2R -4.612 686 23 0.009 925 121 0.000 631 273 0.156 046 663 0.001 032 719 IL1RL2 0.181 334 897 1.198 816 592 1.038 151 643 1.384 346 141 0.013 512 859 TRDC 0.111 556 883 1.118 017 339 1.009 457 521 1.238 251 976 0.032 307 538 注:风险系数coef表示基因对模型风险评分的贡献,负值表示基因与预后呈负相关,正值表示基因与预后呈正相关 -
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