海军军医大学学报  2025, Vol. 46 Issue (1): 79-88   PDF    
MEN1基因与乳腺癌预后及免疫浸润的关系分析
孙晓璐, 黄彦栋, 王莲香, 吴达熙, 黄禾菁     
海军军医大学(第二军医大学)第二附属医院超声科, 上海 200003
摘要: 目的 探讨多发性内分泌肿瘤1型综合征(MEN1)的致病基因MEN1在乳腺癌中的表达及其临床意义。方法 利用肿瘤基因组图谱(TCGA)数据库分析乳腺癌患者MEN1表达水平与临床病理特征的关系,通过Kaplan-Meier生存分析法评估MEN1对乳腺癌预后的影响。采用基因本体(GO)、京都基因与基因组百科全书(KEGG)、基因集富集分析(GSEA)预测乳腺癌中MEN1相关和相互作用基因的功能和相关通路,利用单细胞测序数据库CancerSEA分析MEN1表达与肿瘤生物功能的相关性。采用肿瘤免疫估算资料(TIMER)数据库和单样本基因集富集分析(ssGSEA)研究乳腺癌中免疫细胞浸润水平与MEN1表达的相关性。结果 MEN1在乳腺癌患者中高表达,其表达水平与PAM50分型、围绝经期状态有关(均P<0.05)。Kaplan-Meier生存分析结果显示,MEN1的高表达与较差的临床预后有关(P=0.019)。GO和KEGG富集分析提示MEN1相关和相互作用基因参与组蛋白修饰、组蛋白-赖氨酸甲基化等生物学过程,甲基转移酶复合物、组蛋白甲基转移酶复合物等细胞组分,组蛋白-甲基转移酶活性等分子功能,肿瘤中的转录失调等功能通路。GSEA分析提示高表达MEN1表型涉及囊泡介导转运、补体级联反应、B细胞受体的信号转导、淋巴细胞与非淋巴细胞之间的相互作用、IL的信号转导、免疫系统中的细胞因子信号转导通路。CancerSEA单细胞测序数据分析显示,人乳腺癌细胞MDA-MB-231中MEN1的表达与血管生成呈正相关(P<0.05)。TIMER分析显示,乳腺癌中MEN1表达与巨噬细胞、CD8+ T细胞的浸润水平呈负相关(均P<0.05),与CD4+ T细胞的浸润水平呈正相关(P<0.05)。ssGSEA分析结果提示18种免疫细胞的浸润水平与MEN1表达呈负相关(均P<0.05)。结论 高水平的MEN1预示乳腺癌较短的总生存期,并可能与免疫细胞浸润相关。
关键词: 乳腺肿瘤    多发性内分泌肿瘤1型综合征基因    总生存期    免疫浸润    
Association of MEN1 gene with prognosis and immune infiltration of breast cancer
SUN Xiaolu, HUANG Yandong, WANG Lianxiang, WU Daxi, HUANG Hejing     
Department of Ultrasound, The Second Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200003, China
Abstract: Objective To investigate the expression and clinical significance of multiple endocrine neoplasia type 1 (MEN1) gene in breast cancer. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between MEN1 gene and clinicopathological characteristics of breast cancer. Kaplan-Meier method was used to observe the effect of MEN1 on survival of breast cancer patients. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were used to predict the function and signaling pathways of MEN1 related and interacting genes. Tumor Immune Estimation Resource (TIMER) and single sample gene set enrichment analysis (ssGSEA) were used to investigate the correlation between the level of immune infiltration and MEN1 expression in breast cancer. Results MEN1 was highly expressed in breast cancer patients, and its expression level was related to PAM50 subtype and menopause status (both P < 0.05). Kaplan-Meier survival analysis showed that high MEN1 expression was associated with poor clinical outcome (P=0.019). GO and KEGG enrichment analysis showed that MEN1 related and interacting genes were involved in biological processes (histone modification, histone-lysine methylation), cell components (methyltransferase complex and histone methyltransferase complex), molecular functions (histone-methyltransferase activity), and functional pathways (transcriptional disorders in tumors). GSEA identified that the highly expressed MEN1 phenotype was involved in vesicle-mediated transport, complement cascade, B-cell receptor signaling, lymphocyte-non-lymphocyte interaction, interleukin signaling, and cytokine signaling pathways in the immune system. CancerSEA single cell sequencing results indicated that the expression of MEN1 was positively correlated with angiogenesis in MDA-MB-231 cells (P < 0.05). TIMER analysis found that MEN1 in breast cancer was negatively correlated with the infiltration levels of macrophages and CD8+ T cells, and positively correlated with the infiltration level of CD4+ T cells (all P < 0.05). ssGSEA showed that the infiltration levels of 18 types of immune cells were negatively correlated with MEN1 expression (all P < 0.05). Conclusion High MEN1 level is associated with poor survival and immune infiltration in breast cancer patients.
Key words: breast neoplasms    multiple endocrine neoplasia type 1 gene    overall survival    immune infiltration    

乳腺癌是全球女性发病率和死亡率最高的癌症[1]。乳腺癌的治疗方法包括手术切除、放疗、化疗、内分泌治疗和靶向治疗等,然而在临床实践中,药物不良反应、原发性或获得性耐药等一系列问题仍存在,特别是晚期乳腺癌仍缺乏有效的治疗手段。因此,探索乳腺癌发生的分子机制、寻找生物标志物和新的免疫相关治疗靶点迫在眉睫[2]

多发性内分泌肿瘤1型综合征(multiple endocrine neoplasia type 1,MEN1)是一种罕见的常染色体显性遗传病,由MEN1基因的失活突变引起。MEN1通常编码由610个氨基酸组成的menin蛋白,具有抑制肿瘤的生物功能[3]MEN1与一系列内分泌疾病相关,典型的包括原发性甲状旁腺功能亢进症(primary hyperparathyroidism,PHP)、十二指肠胰腺神经内分泌肿瘤(duodenopancreatic neuroendocrine tumor,DPNET)和垂体腺瘤(pituitary adenoma,PA)[4]MEN1相关肿瘤谱系还包括一些较少见的肿瘤,如小肠神经内分泌肿瘤、肾上腺肿瘤(腺瘤和腺癌)、脂肪瘤、血管纤维瘤、胶原瘤和脑膜瘤。除了这些罕见的肿瘤,一些研究也报告了MEN1患者的乳腺癌患病率高于普通人群[5-6]。在MEN1基因突变的女性中,乳腺癌被认为是MEN1的一部分,临床相关表现在学龄期已经出现,建议10岁开始筛查[7]。然而,乳腺癌是否真的是MEN1相关的恶性肿瘤一直存在争议[6-8]MEN1在乳腺癌中的功能及其与肿瘤免疫的相关性尚不清楚。本研究利用生物信息学工具分析乳腺癌患者MEN1表达水平与临床病理特征及预后的关系,并进一步评估乳腺癌中MEN1表达与其免疫浸润水平的相关性。

1 资料和方法 1.1 数据来源

从肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)数据库下载1 069例乳腺癌组织(其中1例生存信息缺失)和111例乳腺癌旁组织的基因表达数据。通过统一处理TCGA数据库每百万转录本中的转录本数(transcripts per million,TPM)格式的RNAseq数据(https://xenabrowser.net/datapages/),分析MEN1在乳腺癌中的表达[9]。然后对乳腺癌和癌旁正常组织的数据进行具体分析,进一步筛选TCGA中获取的病例的临床病理特征和预后资料。

1.2 MEN1相关和相互作用基因的筛选及功能分析

从基因表达谱交互分析2(Gene Expression Profiling Interactive Analysis 2,GEPIA2)数据库(http://gepia2.cancer-pku.cn/#index)中获得与MEN1表达模式最相似的100个MEN1相关基因。在STRING数据库(https://cn.string-db.org/)中筛选50个MEN1相互作用蛋白质创建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络(设置0.4为最小相互作用阈值)。基于MEN1相关及相互作用基因进行基因本体(Gene Ontology,GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析,进一步探索MEN1的潜在功能。

基于TCGA获得的转录序列,使用基因集富集分析(gene set enrichment analysis,GSEA)识别与MEN1相关的基因集和通路。将基因表达数据分为高表达和低表达MEN1组,使用GSEA对两组进行比较,并通过Broad Institute网站(https://www.broadinstitute.org/)使用R包集群分析器识别潜在的功能[10]。单细胞转录组测序是在单细胞水平分析候选分子潜在功能的关键技术[11]。利用单细胞测序数据库CancerSEA(http://biocc.hrbmu.edu.cn/CancerSEA/)[12],在单细胞水平分析MEN1表达与肿瘤生物功能的相关性。

1.3 免疫细胞浸润分析

免疫细胞在稳态、感染和非感染性干扰中通过清除病原体以保护组织完整性和功能,对肿瘤的临床结局产生一定影响。在乳腺癌中,较高免疫细胞浸润与较好的临床结局和治疗反应相关[13]。为评估肿瘤组织免疫细胞浸润的相对丰度,使用肿瘤免疫估算资料(Tumor Immune Estimation Resource,TIMER)数据库的“Gene”模块,采用Spearman相关分析探讨MEN1与不同免疫细胞的相关性,通过Wilcoxon秩和检验比较MEN1高表达组和低表达组免疫细胞水平的差异。同时进行单样本基因集富集分析(single sample gene set enrichment analysis,ssGSEA),使用“GSVA”(R包)和24种免疫细胞的免疫数据集分析乳腺癌表达谱数据中免疫细胞的浸润水平[13]

1.4 统计学处理

所有统计分析均在R 4.2.1软件中进行。采用Wilcoxon秩和检验对正常组和乳腺癌组的MEN1表达水平进行比较。根据MEN1表达水平的中位数将患者分为MEN1高表达和低表达两组,采用χ2检验对MEN1与乳腺癌临床病理特征的关系进行分析。采用Pearson相关和Spearman相关进行相关性分析。根据最佳截断值将患者分为MEN1高表达和低表达两组,采用Kaplan-Meier生存分析法评估MEN1对乳腺癌预后的影响。使用R包ggplot2 3.3.6绘制所有图形。检验水准(α)为0.05。

2 结果 2.1 MEN1在乳腺癌组织中的表达及其与预后和临床病理特征的关系

利用TCGA数据库分析MEN1在乳腺癌组织和正常乳腺组织中的mRNA表达水平,结果显示乳腺癌组织中MEN1 mRNA的表达水平高于正常组织(P<0.001,图 1A);然后分析MEN1在成对乳腺癌组织和癌旁组织中的表达,结果显示乳腺癌组织MEN1 mRNA水平高于癌旁组织(P<0.001,图 1B)。对从TCGA下载的1 069例乳腺癌患者的MEN1表达与临床病理特征的关系进行分析,结果显示MEN1的表达与病理分期、雌激素受体状态、孕激素受体状态、人表皮生长因子受体2状态、年龄之间无明显关系(均P>0.05),但与PAM50分型、围绝经期状态有关(均P<0.05,表 1)。为证实MEN1在乳腺癌中的作用,采用Kaplan-Meier生存分析研究MEN1 mRNA对乳腺癌患者生存期的影响,结果显示MEN1表达上调与乳腺癌患者较差的总生存期有关(P=0.019,图 2)。

图 1 基于TCGA数据库分析乳腺癌中MEN1 mRNA的表达水平 Fig 1 MEN1 mRNA expression in breast cancer based on TCGA database A: Comparison of MEN1 expression level between 1 069 breast cancer tissues and 111 normal breast tissues; B: MEN1 expression level in 111 matched breast cancer tissues and corresponding normal breast tissues. TCGA: The Cancer Genome Atlas; MEN1: Multiple endocrine neoplasia type 1; TPM: Transcripts per million.

表 1 MEN1高、低表达组乳腺癌患者的临床病理特征比较 Tab 1 Comparison of clinicopathological characteristics between MEN1 high- and low-expression groups in patients with breast cancer   

图 2 MEN1高、低表达组乳腺癌患者的总生存期比较(Kaplan-Meier生存分析) Fig 2 Comparison of overall survival between MEN1 high- and low-expression groups in patients with breast cancer (Kaplan-Meier survival analysis) MEN1: Multiple endocrine neoplasia type 1.

2.2 MEN1相关和相互作用基因功能分析

为了确定MEN1和其他蛋白质在乳腺癌中的作用,从GEPIA2数据库获得1 100例乳腺癌样本中与MEN1表达相关的前100个基因。Pearson相关分析发现,MEN1表达与钙蛋白酶1(calpain-1,CAPN1)、线粒体核糖体蛋白质L49(mitochondrial ribosomal protein L49,MRPL49)、锌指蛋白样蛋白1(zinc finger protein like 1,ZFPL1)呈正相关(r=0.64、0.66、0.64,均P<0.001;图 3A)。使用STRING在线工具获得与MEN1相互作用的50个蛋白质,并绘制PPI网络(图 3B)。取两组预测基因的交集,生成3个共同基因,即宿主细胞因子C1(host cell factor C1,HCFC1)、磷酸酯酶Cβ3(phospholipase C β3,PLCB3)、剪接因子1(splicing factor 1,SF1)(图 3C)。

图 3 MEN1相关和相互作用基因筛选 Fig 3 Screening of MEN1-correlated and interacting genes A: The correlations between MEN1 and top 3 MEN1-correlated genes, including CAPN1, MRPL49, and ZFPL1, obtained from the top 100 MEN1-correlated genes in the TCGA database using the GEPIA2 method; B: PPI networks of the MEN1-binding proteins were created through the STRING tool; C: Venn diagram of the MEN1-correlated and interacting genes. MEN1: Multiple endocrine neoplasia type 1; CAPN1: Calpain-1; MRPL49: Mitochondrial ribosomal protein L49; ZFPL1: Zinc finger protein like 1; TPM: Transcripts per million; TCGA: The Cancer Genome Atlas; GEPIA2: Gene Expression Profiling Interactive Analysis 2; PPI: Protein-protein interaction.

GO分析提示,147个MEN1相关和相互作用基因参与组蛋白修饰、肽基-赖氨酸修饰、组蛋白-赖氨酸甲基化等生物学过程,甲基转移酶复合物、组蛋白甲基转移酶复合物等细胞组分,组蛋白-甲基转移酶活性、蛋白-赖氨酸-N-甲基转移酶活性等分子功能(图 4A)。KEGG分析提示,这些基因大多数与赖氨酸降解、造血系统疾病和肿瘤中的转录失调相关(图 4B)。采用GSEA确定MEN1低表达和高表达数据集之间的信号通路差异,结果显示MEN1与囊泡介导转运、补体级联反应、B细胞受体的信号转导、淋巴细胞与非淋巴细胞之间的相互作用、IL的信号转导、免疫系统中的细胞因子信号转导相关(图 4C)。

图 4 MEN1相关和相互作用基因的富集分析 Fig 4 Enrichment analysis of MEN1-related and interacting genes A, B: The 147 MEN1-interacting and correlated genes were used for the GO (A) and KEGG (B) enrichment analyses; C: GSEA in breast cancer patients with high expression of MEN1 compared with the ones with low expression. MEN1: Multiple endocrine neoplasia type 1; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: Gene set enrichment analysis; BP: Biological process; CC: Cellular component; MF: Molecular function; MLL: Mixed lineage leukemia-fusion protein; NES: Normalized enrichment score; P.adj: Adjusted P value; FDR: False discovery rate.

通过CancerSEA单细胞测序数据探索MEN1在乳腺癌中的功能状态,结果显示,在人乳腺癌细胞MDA-MB-231中,MEN1的表达与血管生成呈正相关(r=0.33,P<0.05)。

2.3 乳腺癌中MEN1表达和免疫浸润水平的关系

利用TIMER数据库分析乳腺癌中MEN1表达与免疫细胞浸润的相关性,结果显示乳腺癌中MEN1的表达与巨噬细胞、CD8+ T细胞的浸润水平呈负相关,与CD4+ T细胞的浸润水平呈正相关,而与B细胞、中性粒细胞和树突状细胞的浸润水平无相关性(图 5A)。进一步分析MEN1高表达和低表达组24种免疫细胞亚型的表达水平,结果显示MEN1高表达组的T细胞、树突状细胞、CD8+ T细胞、B细胞等细胞较低表达组减少(均P<0.01,图 5B)。同时,使用ssGSEA对乳腺癌肿瘤微环境进行富集分析,发现在上述24种免疫细胞中,18种细胞的浸润水平与MEN1表达呈负相关(图 5C)。

图 5 乳腺癌中MEN1表达与免疫浸润水平的关系 Fig 5 Correlation between immune infiltrating level and MEN1 expression in breast cancer A: Relationship between MEN1 expression and immune infiltration level in 1 100 breast cancer using the TIMER database; B: Different levels of 24 subtypes of immune cells in the high and low MEN1 expression groups in the tumor tissue samples; C: Spearman rank correlation method was used to analyze the correlation between immune cell infiltration and MEN1 expression. MEN1: Multiple endocrine neoplasia type 1; TIMER: Tumor Immune Estimation Resource; TPM: Transcripts per million; aDC: Activated dendritic cell; DC: Dendritic cell; iDC: Immature dendritic cell; NK: Natural killer; pDC: Plasmacytoid dendritic cell; Th: Helper T cell; Tcm: Central memory T cell; Tem: Effector memory T cell; TFH: Follicular helper T cell; Tgd: Gamma delta T cell; Treg: Regulatory T cell. **P < 0.01, ***P < 0.001; ns: P > 0.05.

3 讨论

乳腺癌是全球最常见的恶性肿瘤之一。2020年全球乳腺癌病例约有226万例,死亡近68.5万例,是女性癌症死亡的主要原因,其中近三分之二的死亡发生在欠发达地区,较发达地区乳腺癌患者的生存优势在很大程度上归因于早期检测策略、早期诊断和更好地获得有效治疗等方面的综合作用[14]。年龄是乳腺癌发病最重要的危险因素,其他危险因素还包括初潮年龄、绝经年龄、肥胖、饮酒等[15]。乳腺癌的治疗方法有手术、放疗、化疗、内镜治疗和免疫治疗等[16-17],其复发率和死亡率居高不下,多项组学研究发现乳腺癌的肿瘤内和肿瘤间异质性是导致复发或耐药的主要原因[18-19],亟待探寻用于早期筛查的生物标志物和有效的免疫相关治疗靶点。

MEN1基因是公认的肿瘤抑制因子。1997年,有学者首次报道在胰岛素瘤和胃泌素瘤中发现MEN1的突变[20]。随后,对10个非家族性胰腺神经内分泌肿瘤进行的全外显子组测序发现,50%的肿瘤有MEN1失活/错义突变[21]。在非神经内分泌性肿瘤中,MEN1在肺癌、肝癌和造血系统肿瘤中发挥重要的生物学功能。先前的研究报道menin是肝细胞癌相关纤维化发生的关键调节因子,通过增加多梳蛋白(polycomb)介导的组蛋白H3第27位赖氨酸三甲基化和抑制多效蛋白(pleiotrophin,PTN)转录来抑制肺癌[22]、通过调节H3第4位赖氨酸三甲基化与H3第79位赖氨酸二甲基化在混合谱系白血病(mixed lineage leukemia-fusion protein,MLL)的相关融合基因MLL-AF9所致的白血病中起到重要的促瘤作用[23]。在不同组织器官中,MEN1的促癌和抑癌功能并不完全一致,这可能与相应的肿瘤微环境有关。近年来,随着流式细胞技术及细胞特异性基因工程动物的发展,学者们进一步揭示了MEN1在免疫细胞中的功能。menin通过抑制哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)依赖的细胞代谢来维持CD8+ T细胞功能[24]。在小胶质细胞中,menin通过抑制NF-κB/IL-1β通路抑制抑郁症相关的神经性炎症[25]。尽管已有研究表明MEN1通过调节雌激素受体1(estrogen receptor 1,ER1)转录在雌激素受体阳性的乳腺癌细胞中发挥致癌作用[26],但其在乳腺癌发生、发展过程中的作用和机制特别是与肿瘤微环境的关系尚未完全阐明。

本研究发现,与正常组织和癌旁组织相比,MEN1基因在乳腺癌组织中高表达,并与PAM50分型、围绝经期状态及较差的总生存期有关。PAM50亚型是乳腺癌长期生存的独立预后因素[27],因此本研究结果提示将MEN1纳入现有PAM50风险评估工具可能会细化风险分层,为制定个体化治疗策略提供参考。为了解MEN1在乳腺癌中的潜在作用,对MEN1相关和相互作用基因进行GO和KEGG分析,结果表明这些基因参与组蛋白修饰、肽基-赖氨酸修饰、组蛋白-赖氨酸甲基化等生物学过程,甲基转移酶复合物、组蛋白甲基转移酶复合物等细胞成分,组蛋白-甲基转移酶活性、蛋白-赖氨酸-N-甲基转移酶活性等分子功能,赖氨酸降解、造血系统疾病和肿瘤中的转录失调等功能通路。这提示MEN1在乳腺癌中参与多种转移酶复合物的合成及活性调节,进一步揭示了其在表观遗传水平的调控机制。单细胞测序分析结果提示MEN1表达与血管生成呈正相关,GSEA分析进一步提示MEN1在乳腺癌中发挥作用的潜在分子机制可能与囊泡介导转运、补体级联反应、B细胞受体的信号转导、淋巴细胞与非淋巴细胞之间的相互作用、IL的信号转导、免疫系统中的细胞因子信号转导等相关。

近年来,免疫细胞浸润在癌症发生、发展中的作用越来越受到关注[28]。免疫疗法在多种肿瘤的治疗中取得了良好的临床效果。浸润的免疫细胞在调节癌细胞识别和肿瘤生长中发挥重要作用[29-30]。免疫细胞在稳态、感染和非感染性干扰中通过清除病原体以保护组织完整性和功能,对肿瘤的临床结局产生一定影响。在乳腺癌中,较高免疫细胞浸润与较好临床结局以及治疗反应相关[31]。B细胞最广为人知的功能是产生抗体,如IgM、IgG、IgE和IgA[32]。耗竭效应B细胞和T细胞可使肿瘤细胞逃避免疫监视,从而降低肿瘤患者的总生存期[33]。在乳腺癌和其他实体瘤患者中,肿瘤浸润淋巴细胞的存在与良好结局相关。T细胞在肿瘤浸润淋巴细胞中占相当大的比例,目前的证据表明,CD8+ T细胞是良好临床预后的关键决定因素[34]。树突状细胞能够将抗原交叉提呈给CD4+和CD8+ T细胞,从而激活T细胞攻击肿瘤细胞[35]。巨噬细胞是肿瘤微环境的重要组成部分,通过创造免疫抑制微环境在肿瘤发展和耐药中发挥重要作用[36]MEN1在乳腺癌中的表达与CD8+ T细胞、巨噬细胞呈负相关,与CD4+ T细胞呈正相关,提示MEN1可能在调节乳腺癌免疫微环境中发挥重要作用。本研究发现MEN1的表达与B细胞、巨噬细胞、中性粒细胞等免疫细胞浸润密切相关,提示MEN1可能成为免疫治疗的有效靶点,为肿瘤患者的临床治疗提供了新的思路。

综上所述,本研究通过生物信息学分析探索了MEN1在乳腺癌中的表达水平,以及MEN1对乳腺癌临床预后及免疫调节的影响,提示MEN1可能是一种潜在的与乳腺癌患者预后和免疫相关的生物标志物。但MEN1影响乳腺癌肿瘤免疫微环境和肿瘤进展的具体机制尚不明确,需要进一步开展基础研究和临床试验,以全面阐明MEN1在乳腺癌中的生物学作用。

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