Supplementary Material for: Effects of Fibronectin 1 on Cell Proliferation, Senescence and Apoptosis of Human Glioma Cells Through the PI3K/AKT Signaling Pathway

Background/Aims: The current study aimed to investigate the role by which fibronectin 1 (FN1) influences the cell cycle, senescence and apoptosis in human glioma cells through the PI3K/ AKT signaling pathway. Methods: Differentially expressed genes (DEGs) were identified based on gene expression data (GSE12657, GSE15824 and GSE45921 datasets) and probe annotation files from Gene Expression Omnibus. The DEGs were identified in connection with gene ontology (GO) enrichment analysis and with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The positive expression of the FN1 protein was detected by immunohistochemistry. The glioma cell lines U251 and T98G were selected and assigned into blank, negative control (NC) and siRNA-FN1 groups. A dual luciferase reporter gene assay was used to investigate the effects of FN1 on transcriptional activity through the PI3K/AKT signaling pathway. An MTT assay was applied for the detection of cell proliferation, while flow cytometry was employed for cell cycle stage and cellular apoptosis detection. β-galactosidase staining was utilized to detect cellular senescence, a scratch test was applied to evaluate cell migration, and a transwell assay was used to analyze cell invasion. Western blotting and qRT-PCR methods were used to detect the protein and mRNA expression levels, respectively, of the FN1 gene and the related genes in the PI3K/AKT pathway (PI3K, AKT and PTEN), the cell cycle (pRb, CDK4 and Cyclin D1) and cell senescence (p16 and p21) among the collected tissues and cells. Results: GSE12657 profiling revealed FN1 to be the most upregulated gene in glioma. Regarding the GSE12657 and GSE15824 datasets, FN1 gene expression was higher in glioma tissues than in normal tissues. GO enrichment analysis and KEGG pathway enrichment analysis indicated that FN1 is involved in the synthesis of extracellular matrix (ECM) components and the PI3K/AKT signaling pathway. Verification was provided, indicating the role played by the FN1 gene in the regulation of the PI3K/AKT signaling pathway, as silencing the FN1 gene was found to inhibit cell proliferation, promote cell apoptosis and senescence, and reduce migration and invasion through the down-regulation of FN1 gene expression and disruption of the PI3K-AKT signaling pathway. Conclusion: The findings of this study provide evidence highlighting the prominent role played by FN1 in stimulating glioma growth, invasion, and survival through the activation of the PI3K/AKT signaling pathway.