Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

癌症治疗是抑制乳腺癌干细胞（BCSC）途径的战略措施。柚皮素是一种柑橘类黄酮，被发现可以增加乳腺癌细胞对化疗药物的敏感性。本研究采用乳腺癌（乳腺球）生物信息学研究和3D肿瘤球体外建模，旨在探索柚皮素（PTTNs）抑制BCSCs的潜在治疗靶点。生物信息学分析确定了直接靶蛋白 (DTP)、间接靶蛋白 (ITP)、柚皮素介导蛋白 (NMP)、BCSC 调节基因和 PTTN。进一步分析 PTTN 的基因本体、京都基因和基因组百科全书 (KEGG) 途径富集、蛋白质-蛋白质相互作用 (PPI) 网络和中心蛋白质选择。微球在无血清培养基中培养。通过基于 MTT 的细胞毒性、乳腺球形成潜力 (MFP)、集落形成、划痕伤口愈合测定以及基于流式细胞术的细胞周期分析和细胞凋亡测定来测量柚皮素的作用。使用实时定量聚合酶链反应（q-RT PCR）进行基因表达分析。生物信息学分析显示p53和雌激素受体α（ERα）为PTTN，KEGG通路富集分析显示TGF-β和Wnt/β-catenin通路受PTTN调节。柚皮素表现出细胞毒性，并抑制乳腺球和集落的形成、迁移以及乳腺球中上皮到间质的转变。肿瘤抑制因子 P53 和 ERα 的 mRNA 在乳腺球中下调，但在柚皮素处理后显着上调。通过调节 P53 和 ERα mRNA，柚皮素具有抑制 BCSC 的潜力。进一步研究柚皮素在 BCSC 中的分子机制和制剂将有利于其作为 BCSC 靶向药物的开发。