Effects of Huaier extract and autophagy factors on cholangiocarcinoma

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.