Exploring the Mechanism of Polyphyllin Ⅰ on Inhibition of Glioblastoma Invasion and Migration Based on TGF-β1-mediated Epithelial-mesenchymal Transition
Objective Bioinformatics and experimental validation were used to explore the mechanism of action of polyphyllin Ⅰ on regulating epithelial-mesenchymal transition (EMT) to inhibit the invasion and migration of glioblastoma (GBM). Methods Firstly,differentially expressed genes (DEGs) of glioblastoma were screened by The Cancer Genome Atlas Program(TCGA)database,and then they were used for enrichment analysis to explore the pathogenesis-related mechanism. Subsequently,the predicted targets of polyphyllin Ⅰ were taken to intersect with the DEGs of the disease to construct a protein interaction network. The key targets for the therapeutic effects of the drug were screened and used for enrichment analysis. Furthermore,molecular docking was applied for validation to clarify the potential molecular mechanism of polyphyllin Ⅰ for the treatment of glioblastoma,which was then validated by in vitro experiments based on the above bioinformatics results. After the intervention of concentration-gradient polyphyllin Ⅰ against human glioblastoma cell lines LN229 and U251,cell activity was detected by Cell Counting Kit-8(CCK-8),invasive ability of glioblastoma cells was detected by Transwell assay,and migratory ability of glioblastoma cells was detected by scratch assay. Immunoblotting assay was performed to detect protein expression of transforming growth factor β1 (TGFβ1),cadherin 1 (CDH1),cadherin 2 (CDH2) and vimentin (VIM). Finally,differences in the expression of CDH2,TGFβ1,and VIM in glioblastoma and normal tissues were analyzed by Gene Expression Profiling Interaction Analysis database (GEPIA) to predict their effects on patient survival. Results Seven key targets,including CDH1,CDH2 and TGFβ1,were screened based on bioinformatics analysis,and enrichment analysis revealed that the above targets were closely related to the EMT biological process and TGF β signaling pathway. The cell viability was significantly reduced after the intervention of concentration-gradient polyphyllin Ⅰ against LN229 and U251 (P<0.05,P<0.01). Compared with the control group,polyphyllin Ⅰ significantly inhibited the invasion(P<0.01) and migration ability(P<0.05,P<0.01)of LN229 and U251 cells,up-regulated the protein expression of CDH1 and down-regulated the protein expression of TGFβ1,CDH2 and VIM (P<0.05,P<0.01) in a dose-dependent effect (P<0.01). GEPIA analysis revealed that the expression of CDH2,TGFβ1 and VIM in glioblastoma tissues were significantly higher than those in normal tissues(P<0.05),and the high expression of TGFβ1 and VIM might be the key factors affecting the disease-free survival of patients (P<0.05). Conclusion Polyphyllin Ⅰ can inhibit the invasion and migration ability of LN229 and U251 cells through TGFβ1-mediated EMT process. This study provides a new research direction and evidence support for the clinical precision treatment and the development of traditional Chinese medicine small molecules.
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