背景:据报道,免疫球蛋白λ(Igλ)在许多正常和肿瘤组织和细胞中表达。然而,肿瘤来源的Igλ的功能和临床意义尚不清楚。
方法:用癌症基因组图谱(TCGA)检测了免疫球蛋白Lambda常数(IGLC)在宫颈鳞状细胞癌和宫颈腺癌(CESC)中的差异表达。基因型-组织表达(GTEx),和人类蛋白质图谱(HPA)数据库。通过基于TCGA数据库的生物信息学分析探索IGLC对患者临床表型和预后的影响。我们使用基于TCGA和GTEx数据库的生物信息学分析来阐明IGLC表达之间的相关性,免疫调节剂表达,肿瘤干性,肿瘤浸润免疫细胞的浸润评分。免疫共沉淀(Co-IP)和银染结合液相色谱-串联质谱(LC-MS/MS)来获得潜在的肿瘤来源的Igλ相互作用蛋白。通过LC-MS/MS鉴定的候选蛋白的功能注释在数据库中进行注释,可视化和集成发现(DAVID)。基于TCGA对CESC和正常宫颈组织中的7种IGLC进行了生物信息学分析,GTEx,和基因表达谱交互式分析2(GEPIA2)数据库。在用于检索相互作用基因/蛋白质(STRING)数据库的搜索工具中,基于肿瘤来源的Igλ相互作用蛋白分析蛋白质-蛋白质相互作用(PPI)网络。免疫组织化学(IHC)用于验证IGLC在CESC中的表达。
结果:我们发现大多数IGLCs(IGLC1、IGLC2、IGLC3、IGLC4、IGLC5、IGLC6和IGLC7)在CESC组织中表达上调,与正常宫颈组织相比。IGLC5和IGLC7在不同病理转移(M)中的表达差异有统计学意义,肿瘤之一,节点,和转移(TNM)分期系统,CESC的类别。除无病间隔(DFI)外,4IGLC(IGLC1、IGLC2、IGLC3和IGLC7)表达水平与患者总生存期(OS)呈正相关,疾病特异性生存率(DSS),CESC组织中的无进展间隔(PFI)。5IGLC(IGLC1,IGLC2,IGLC3,IGLC6和IGLC7)的表达分别与CESC组织中大多数免疫调节剂的表达呈正相关。CESC组织中4种IGLC1、IGLC2、IGLC3、IGLC7的表达与肿瘤干性呈负相关。除IGLC4,IGLC5和IGLC7外,4个IGLC(IGLC1,IGLC2,IGLC3和IGLC6)的表达与6个肿瘤浸润性免疫细胞(B细胞,T细胞CD4,T细胞CD8,中性粒细胞,巨噬细胞,和DC)。在分析了上述肿瘤来源的Igλ的生物信息学数据后,Co-IP和LC-MS/MS用于确认4种蛋白质(RPL7,RPS3,H1-5和H1-6)可能与宫颈癌细胞中的肿瘤来源的Igλ相互作用。这些候选蛋白质的功能分析显示它们与许多蛋白质相互作用并且参与各种细胞生物学过程。最后,IHC用于进一步证实上述生物信息学结果,结果表明,宫颈腺癌和宫颈鳞癌中的Igλ表达水平高于正常宫颈组织。
结论:本研究基于生物信息学分析,全面研究了肿瘤源性Igλ及其相互作用蛋白的功能,以及Igλ作为CESC预后和治疗标志物的潜在价值。为CESC治疗提供新的方向和证据。
BACKGROUND: Immunoglobulin lambda (Igλ) has been reported to be expressed in many normal and tumor tissues and cells. However, the function and clinical significance of tumor-derived Igλ remain unclear.
METHODS: The differential expressions of Immunoglobulin Lambda Constants (IGLCs) in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) were examined with The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) databases. The effects of IGLCs on patient clinical phenotypes and prognosis were explored via bioinformatics analyses based on the TCGA databases. We used the bioinformatics analyses based on the TCGA and GTEx databases to elucidate the correlations among IGLC expressions, immunomodulator expressions, tumor stemness, and infiltration scores of tumor infiltrating immune cells. Co-immunoprecipitation (Co-IP) and silver staining combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to obtain potential tumor-derived Igλ-interacting proteins. Functional annotation of candidate proteins identified by LC-MS/MS was performed in Database for Annotation, Visualization and Integrated Discovery (DAVID). The bioinformatics analyses of 7 IGLCs in CESC and normal cervical tissues were performed based on TCGA, GTEx, and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) databases. Protein-protein interaction (PPI) network was analyzed based on tumor-derived Igλ-interacting proteins in Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Immunohistochemistry (IHC) was used to validate the expressions of IGLCs in CESC.
RESULTS: We found that the expressions of the majority of IGLCs (IGLC1, IGLC2, IGLC3, IGLC4, IGLC5, IGLC6, and IGLC7) were upregulated in CESC tissues, compared with those in normal cervical tissues. The expressions of IGLC5 and IGLC7 had significant difference in different pathologic metastasis (M), one of tumor, node, and metastasis (TNM) staging system, categories of CESC. Except for disease-free interval (DFI), 4 IGLC (IGLC1, IGLC2, IGLC3, and IGLC7) expression levels were positively associated with patient overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) respectively in CESC tissues. 5 IGLC (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7) expressions were positively correlated with the expressions of a majority of immunomodulators respectively in CESC tissues. Tumor stemness was negatively correlated with the expressions of 4 IGLCs (IGLC1, IGLC2, IGLC3, and IGLC7) respectively in CESC tissues. Except for IGLC4, IGLC5, and IGLC7, 4 IGLC (IGLC1, IGLC2, IGLC3, and IGLC6) expressions were positively correlated with infiltration scores of 6 tumor-infiltrating immune cells (B cell, T cell CD4, T cell CD8, neutrophil, macrophage, and DC). After analyses of the above bioinformatics data of tumor-derived Igλ, Co-IP and LC-MS/MS were used to confirm that 4 proteins (RPL7, RPS3, H1-5, and H1-6) might interact with tumor-derived Igλ in cervical cancer cells. Functional analyses of these candidate proteins showed that they interacted with many proteins and were involved in various cellular biological processes. Finally, IHC was used to further confirm the above bioinformatics results, it was indicated that the expression level of Igλ in cervical adenocarcinoma and cervical squamous cell carcinoma was higher than that in normal cervical tissue.
CONCLUSIONS: This study comprehensively investigated the functions of tumor-derived Igλ and its interacting proteins based on bioinformatics analysis and the potential value of Igλ as a prognostic and therapeutic marker for CESC, providing new direction and evidence for CESC therapy.