[Mechanism of betulinic acid against colorectal cancer based on bioinformatics and experimental validation]

This study explored the therapeutic potential and underlying mechanisms of betulinic acid(BA) in the treatment of colorectal cancer(CRC) through integrated bioinformatics analysis and in vitro experimental validation. Potential targets of BA against CRC were identified by mining multiple public databases, including BATMAN-TCM, PharmMapper, DrugBank, and GeneCards. A total of 100 overlapping targets were identified. Protein-protein interaction(PPI) network was constructed to screen for core targets. Differentially expressed genes(DEGs) in CRC patients were obtained from the cancer genome atlas(TCGA), and single-cell RNA sequencing analysis was employed to identify the specific cell clusters potentially affected by BA. Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed on the common targets to clarify the potential signaling pathways and biological processes. Core targets were further validated through molecular docking with BA. A methyltransferase-like 3(METTL3) knockdown model was established using HCT116 colorectal cancer cells. The cells were treated with varying concentrations of BA(9.375, 18.75, 37.5, 75, and 150 μmol·L~(-1)). Cell viability was assessed using CCK-8 assay. Additionally, wound healing assays were used to assess cell migration ability, while Transwell assays were conducted to evaluate both migration and invasion ability. Western blot analysis was performed to measure the expression levels of METTL3, as well as peroxisome proliferator-activated receptors(PPAR)γ and PPARα. Network pharmacology analysis revealed that the therapeutic effects of BA on CRC were primarily associated with 100 therapeutic targets and 26 signaling pathways, with PPAR signaling pathway identified as a key pathway. Molecular docking demonstrated that BA exhibited a strong binding affinity to the core targets. In vitro experiments confirmed that BA significantly inhibited the proliferation, migration, and invasion of HCT116 cells. Western blot indicated that BA treatment downregulated METTL3 expression and reduced the expression of PPARγ and PPARα compared to the control group. These findings suggest that BA may inhibit the proliferation, migration, and invasion of CRC cells by down-regulating METTL3 expression, thereby reducing the m~6A methylation level of PPARγ and PPARα mRNA, leading to decreased mRNA stability and translational efficiency, and ultimately modulating the PPAR signaling pathway.