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

<b><i>Background/Aims:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.

**背景与目的：** 本研究旨在探讨纤连蛋白1（fibronectin 1, FN1）通过PI3K/AKT信号通路对人脑胶质瘤细胞周期、细胞衰老及细胞凋亡的调控作用。**方法：** 基于基因表达综合数据库（Gene Expression Omnibus, GEO）的基因表达数据（GSE12657、GSE15824及GSE45921数据集）及探针注释文件，鉴定差异表达基因（differentially expressed genes, DEGs）；结合基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析完成差异表达基因的筛选与注释。采用免疫组化法检测FN1蛋白的阳性表达情况。选取人脑胶质瘤细胞系U251与T98G，分为空白对照组、阴性对照（negative control, NC）组及siRNA-FN1干扰组。利用双荧光素酶报告基因实验，探究FN1通过PI3K/AKT信号通路对转录活性的影响。采用MTT法检测细胞增殖能力，流式细胞术检测细胞周期时相与细胞凋亡水平；通过β-半乳糖苷酶染色检测细胞衰老状态，划痕实验评估细胞迁移能力，Transwell实验分析细胞侵袭能力。采用蛋白质印迹法（Western blotting）与实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR），分别检测收集的组织与细胞样本中FN1基因、PI3K/AKT通路相关基因（PI3K、AKT及PTEN）、细胞周期相关基因（pRb、CDK4及Cyclin D1）以及细胞衰老相关基因（p16、p21）的蛋白与mRNA表达水平。**结果：** GSE12657数据集分析显示，FN1为胶质瘤组织中上调幅度最高的基因；对比GSE12657与GSE15824数据集，胶质瘤组织内FN1基因表达水平显著高于正常脑组织。GO富集分析与KEGG通路富集分析结果表明，FN1参与细胞外基质（extracellular matrix, ECM）组分合成及PI3K/AKT信号通路调控。验证实验证实，FN1基因可调控PI3K/AKT信号通路：敲低FN1基因表达可通过下调FN1基因转录水平、阻断PI3K-AKT信号通路，进而抑制细胞增殖、促进细胞凋亡与衰老，并降低细胞迁移与侵袭能力。**结论：** 本研究结果表明，FN1可通过激活PI3K/AKT信号通路，在胶质瘤的生长、侵袭及存活过程中发挥关键促进作用。