2020年乳腺癌已成为全球死亡率和发病率最高的癌症，而三阴性乳腺癌（TNBC）由于同时缺乏常见的三种受体，导致它对女性的威胁极强，癌症转移到远端组织发生率高且术后易复发，这类分型约占所有乳腺癌15%左右，年轻女性多发。如何有效治疗并改善患者预后成为近年来的研究热点，但多种靶向药仍处于在研阶段。肿瘤干细胞（CSCs）近年来被认为可能是导致癌症转移、侵袭力强的原因，目前已证实乳腺癌中存有肿瘤干细胞。对于侵袭力极强的TNBC，本研究希望能通过探索三阴性乳腺癌肿瘤干细胞（TNBC-CSCs）与TNBC细胞之间的差异表达蛋白，得到通过CSCs影响TNBC预后的生物标志物，为该类肿瘤靶向治疗提供研究基础。通过细胞培养我们首先培养出TNBC-CSCs，并通过碱性磷酸酶检测、免疫荧光、干性基因表达量检测鉴定了我们体外诱导的肿瘤干细胞。随后，通过两种细胞的蛋白质组测序（iTRAQ法），得出这两类细胞共有988个表达升高和1087个表达降低的差异表达蛋白。对这些细胞进行聚类分析、功能富集、通路分析，我们发现整合素家族众多蛋白是TNBC与TNBC-CSCs的差异表达蛋白，在ECM受体相互作用通路显著富集。蛋白免疫印迹实验证实富集的整合素家族蛋白在TNBC-CSCs中显著上调，HPA数据库证实这些蛋白在乳腺癌临床样本中表达量具有相同趋势。预后及ROC分析证实这些蛋白导致TNBC患者预后不良。ITGB5在TNBC中的作用尚未被研究。我们用siRNA敲低TNBC-CSCs中ITGB5并发现细胞的迁移能力被抑制，而过表达TNBC-CSCs中的ITGB5又导致细胞干性基因表达量升高。我们通过ITGB5与免疫细胞的相关性分析发现ITGB5可能通过调节巨噬细胞极化影响肿瘤进展，而对ITGB5相关性高的150个基因功能分析得出它们主要集中在ECM受体相互作用、细胞粘附及PI3K-AKT通路。上述结果表明ITGB5可能是通过TNBC-CSCs影响TNBC预后的生物标志物。
Breast cancer has become the cancer with the highest incidence and mortality worldwide in 2020. Due to a lack of three main receptors, ER, PR and HER-2, triple-negative breast cancer (TNBC) has high recurrence and metastasis rates. And it accounts for about 15% of all breast cancers. How to effectively treat and improve the prognosis of patients has become a research hotspot in recent years, but a variety of targeted drugs are still under development. In recent years, Cancer stem cells (CSCs) are responsible for tumor metastasis and invasion, and CSCs have been proved to be present in breast cancer. For highly invasive TNBC, we want to explore the differentially expressed proteins between triple-negative breast cancer stem cells (TNBC-CSCs) and TNBC and obtain biomarkers that can influence the prognosis of triple-negative breast cancer via CSCs.Specifically, TNBC-CSCs were cultured by cell suspension method and identified by alkaline phosphatase detection, immunofluorescence assay, quantitative real time PCR (qRT-PCR) and western blot to detect expression of stemness biomarkers. Subsequently, a total of 988 higher expressed proteins and 1087 lower expressed proteins were identified by isobaric tag for relative and absolute quantitation (iTRAQ) of the two types of cells. Cluster analysis, GO function enrichment analysis and KEGG pathway enrichment analysis were performed to revealed that many proteins in integrin family were differentially expressed proteins of TNBC and TNBC-CSCs, and significantly enriched in the ECM receptor interaction pathway. Western blot confirmed these proteins from integrin family were significantly up-regulated in TNBC-CSCs, and Human Protein Atlas Database (HPA) confirmed that the expression of proteins in clinical samples of breast cancer have the same trend. KM survival analysis and time-dependent receiver operating characteristic (ROC) curve analysis revealed that these proteins are related to poor prognosis in TNBC patients.The role of ITGB5 from integrin family in TNBC has not been studied. We knocked down ITGB5 in TNBC-CSCs with siRNA and found that the migration ability of TNBC-CSCs was inhibited, while overexpression of ITGB5 in TNBC-CSCs led to higher expressed of stemness genes. By correlation analysis of ITGB5 and immune cells, we found that ITGB5 may affect tumor progression by regulating macrophage polarization, while functional enrichment analysis of 150 genes with high correlation of ITGB5 showed that these genes were mainly correlated with ECM receptor interaction pathway, cell adhesion pathway and PI3K-AKT pathway. Our findings suggested that ITGB5 was a potential biomarker affecting the prognosis of TNBC via TNBC-CSCs.
