Yang Chenrui, Wang Yanliang, Zhang Yanzhong, Liu Yajuan, Wu Xiaoyong
Department of General Surgery, Danzhou People's Hospital, Danzhou City, Hainan Province, China.
Department of Hepatobiliary Surgery, Danzhou People's Hospital, Danzhou City, Hainan Province, China.
Medicine (Baltimore). 2025 Jul 18;104(29):e43421. doi: 10.1097/MD.0000000000043421.
This study aims to investigate the mechanisms by which Huaier extract and autophagy-related factors influence biological functions such as survival and proliferation in cholangiocarcinoma cells. HUCCT1 and QBC939 cholangiocarcinoma cell lines were treated with varying concentrations of Huaier extract (0, 20, 40, and 100 mg/mL) for 24 hours. Cell viability and proliferation were assessed using CCK8 and EdU assays. Flow cytometry was employed to analyze cell cycle distribution and apoptosis. Transwell assays evaluated cell migration and invasion capabilities. Western blotting analyzed protein expression levels of P53, phosphorylated P53, AKT, phosphorylated AKT, ribosomal protein S6, Bcl-2, and Bax in control and high-dose Huaier-treated groups. To explore the role of autophagy in cholangiocarcinoma, gene expression datasets were retrieved from the Gene Expression Omnibus for differential expression analysis. Weighted gene co-expression network analysis identified key gene modules. Protein-protein interaction networks and functional enrichment analyses were conducted, with gene expression heatmaps generated. The comparative toxicogenomics database was used to associate core genes with diseases, while TargetScan predicted microRNAs regulating differentially expressed genes. In HUCCT1 cells, Huaier treatment reduced viability and proliferation in a dose-dependent manner and increased apoptosis. High-dose Huaier significantly decreased Bcl-2, RPS6, AKT, and phosphorylated AKT protein levels. Similarly, in QBC939 cells, Huaier reduced viability, proliferation, migration, and invasion, while promoting apoptosis. High-dose treatment notably decreased RPS6 expression and significantly increased P53 and phosphorylated P53 levels. Bioinformatics analysis identified 4248 differentially expressed genes in cholangiocarcinoma samples. Three core autophagy-related genes (BECN1, ATG7, and DRAM1) were pinpointed. These genes were enriched in autophagy processes, cytoplasmic functions, autophagosome membrane formation, the PI3K-Akt signaling pathway, and apoptosis, with elevated expression in tumor samples. Comparative toxicogenomics database analysis linked these core genes to cholangiocarcinoma, inflammation, necrosis, and proliferation. Huaier extract and autophagy factors Beclin 1, autophagy-related gene 7, and damage-regulated autophagy modulator 1 play significant roles in regulating the growth and proliferation of cholangiocarcinoma cells, highlighting potential therapeutic targets.
本研究旨在探讨槐耳提取物和自噬相关因子影响胆管癌细胞生存和增殖等生物学功能的机制。将HUCCT1和QBC939胆管癌细胞系用不同浓度的槐耳提取物（0、20、40和100mg/mL）处理24小时。使用CCK8和EdU检测评估细胞活力和增殖。采用流式细胞术分析细胞周期分布和凋亡情况。Transwell检测评估细胞迁移和侵袭能力。蛋白质印迹法分析对照和高剂量槐耳处理组中P53、磷酸化P53、AKT、磷酸化AKT、核糖体蛋白S6、Bcl-2和Bax的蛋白表达水平。为了探究自噬在胆管癌中的作用，从基因表达综合数据库检索基因表达数据集进行差异表达分析。加权基因共表达网络分析确定关键基因模块。进行蛋白质-蛋白质相互作用网络和功能富集分析，并生成基因表达热图。利用比较毒理基因组学数据库将核心基因与疾病关联起来，同时TargetScan预测调控差异表达基因的微小RNA。在HUCCT1细胞中，槐耳处理以剂量依赖方式降低细胞活力和增殖，并增加凋亡。高剂量槐耳显著降低Bcl-2、RPS6、AKT和磷酸化AKT蛋白水平。同样，在QBC939细胞中，槐耳降低细胞活力、增殖、迁移和侵袭，同时促进凋亡。高剂量处理显著降低RPS6表达，并显著增加P53和磷酸化P53水平。生物信息学分析确定胆管癌样本中有4248个差异表达基因。确定了三个核心自噬相关基因（BECN1、ATG7和DRAM1）。这些基因在自噬过程、细胞质功能、自噬体膜形成、PI3K-Akt信号通路和凋亡中富集，在肿瘤样本中表达升高。比较毒理基因组学数据库分析将这些核心基因与胆管癌、炎症、坏死和增殖联系起来。槐耳提取物和自噬因子Beclin 1、自噬相关基因7和损伤调节自噬调节剂1在调节胆管癌细胞的生长和增殖中发挥重要作用，凸显了潜在的治疗靶点。
