卵巢粒层细胞瘤与纤维卵泡膜细胞瘤蛋白质组学研究

Proteomic study on ovary granulosa cell tumor and ovarian fibrothecoma

目的:卵巢粒层细胞瘤(ovary granulosa cell tumor,OGCT)与纤维卵泡膜细胞瘤(ovarian fibrothecoma,OF)是卵巢性索间质肿瘤较为常见的类型。由于二者均含有卵泡膜细胞且OGCT呈弥漫性生长模式时与富于细胞的OF和黄素化的OF组织学形态具有相似性,造成二者在病理诊断过程中鉴别困难。本文旨在通过探究OGCT与OF蛋白质组学差异、筛选二者间差异表达蛋白质(differentially expressed proteins,DEPs),发现鉴别诊断二者的生物学标志物。方法:选取5例OGCT和5例OF患者的新鲜肿瘤样本组织,采用蛋白质消化、肽段纯化技术及液相色谱-质谱/质谱分析进行蛋白质定量。运用软件对OGCT和OF的蛋白质进行DEPs分析;对DEPs进行基因本体(gene ontology,GO)和京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,并在生物过程(bioprocess,BP)、分子功能(molecular function,MF)和细胞组分(cell component,CC)方面对蛋白质/基因进行功能注释,探讨DEPs显著富集的信号通路;通过检索基因/蛋白质相互作用(search tool for the retrieval of interacting genes/proteins,STRING)数据库获得筛选出的DEPs相互作用信息,构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络。选取26例OGCT和28例OF石蜡包埋组织,通过组织芯片技术和免疫组织化学染色法验证筛选出的DEPs在OGCT和OF中的表达情况。结果:共筛选出24个OGCT与OF显著DEPs,其中13个蛋白质在OGCT中表达量显著上调,11个蛋白质在OF中表达量显著上调。GO富集分析结果显示:富集到BP上的通路包括细胞外基质(extracellular matrix,ECM)处理过程,细胞外结构组织,有机酸、羧酸、脂肪酸和单羧酸分解代谢过程;富集到CC上的通路包括含胶原蛋白的ECM、内质网腔和线粒体基质;富集到MF上的通路包括硫化物结合、ECM结构成分、酰胺结合、胶原结合、转移酶活性和转移酰基。KEGG富集分析结果显示:上调的DEPs主要富集于缬氨酸、亮氨酸和异亮氨酸降解途径;下调的DEPs主要富集于先天性谷胱甘肽代谢缺陷症、朊病毒疾病、花生四烯酸代谢途径、补体系统、细胞色素P450对异种生物的代谢作用和药物代谢-细胞色素P450。PPI结果显示24个DEPs与多种蛋白质存在相互作用关系。组织芯片免疫组织化学染色显示:PLOD3在OGCT中高表达,在OF中低表达,与富集分析结果一致。结论:通过蛋白质组学技术和生物信息学分析方法共筛选出24个DEPs,经免疫组织化学验证,PLOD3在OGCT与OF鉴别诊断中具有一定的临床应用价值,有望成为OGCT与OF鉴别诊断的生物学标志物。

Objective:Ovary granulosa cell tumor(OGCT)and ovarian fibrothecoma(OF)are relatively common types of ovarian sex cord-stromal tumors.Both contain follicular membrane cells,and when OGCT exhibits a diffuse growth pattern,it shares similarities in histological morphology with cell-rich OF and luteinized OF,causing difficulty in pathological diagnosis.This study aims to explore the proteomic differences,and screen for differentially expressed proteins(DEPs)between them,and find biomarkers for distinguishing between the OGCT and OF.Methods:Fresh tumor tissue samples from 5 patients with OGCT and 5 patients with OF were selected.Protein quantification was performed using protein digestion,peptide purification,and liquid chromatography-mass spectrometry/mass spectrometry analysis(LC-MS/MS).Software was used to analyze DEPs in OGCT and OF proteins.The gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on DEPs,and functional annotations of proteins/genes were conducted in terms of biological processes(BP),molecular function(MF),and cell component(CC)to explore the signaling pathways significantly enriched by DEPs.Interaction information of selected DEPs was obtained from the search tool for the retrieval of interacting genes/proteins(STRING)database to construct a protein-protein interaction(PPI)network.Tissue chip technology and immunohistochemical staining were used to verify the expression of selected DEPs in OGCT and OF in 26 OGCT and 28 OF paraffin-embedded tissues.Results:A total of 24 significantly DEPs between OGCT and OF were identified,with 13 proteins up-regulated in OGCT and 11 proteins up-regulated in OF.GO enrichment analysis showed that pathways enriched in BP included extracellular matrix(ECM)organization processes,extracellular structural organization,organic acid,carboxylic acid,fatty acid,and monocarboxylic acid catabolic processes.Pathways enriched in CC included ECM containing collagen,endoplasmic reticulum lumen,and mitochondrial matrix.Pathways enriched in MF included sulfide compound binding,ECM structural constituents,amide binding,collagen binding,transferase activity,and acyltransferase.KEGG enrichment analysis showed that upregulated DEPs were mainly enriched in valine,leucine,and isoleucine degradation pathways,while downregulated DEPs were mainly enriched in defects in congenital glutathione metabolism,deficiency,prion disease,arachidonic acid metabolic pathway,complement system,cytochrome P450 metabolism on heterogeneic organisms,and drug metabolism-cytochrome P450.PPI results showed interactions among the 24 DEPs and multiple proteins immunohistochemical staining of tissue chips showed that PLOD3 was highly expressed in OGCT and low in OF,consistent with the enrichment analysis results.Conclusion:Through proteomic technology and bioinformatics analysis methods,a total of 24 DEPs are selected,and through immunohistochemical validation,the detection of PLOD3 has potential clinical value in distinguishing between OGCT and OF,and it is expected to become a biological marker for the differential diagnosis of OGCT and OF.