Liao Yu-Xiang, Zhang Zhi-Ping, Zhao Jie, Liu Jing-Ping
Cell Physiol Biochem. 2018;48(3):1382-1396. doi: 10.1159/000492096. Epub 2018 Jul 26.
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.
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.
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.
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/目的：本研究旨在探讨纤连蛋白1（FN1）通过PI3K/AKT信号通路影响人胶质瘤细胞的细胞周期、衰老和凋亡的作用机制。
基于基因表达综合数据库（GSE12657、GSE15824和GSE45921数据集）及探针注释文件，鉴定差异表达基因（DEGs）。通过基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）通路富集分析对DEGs进行鉴定。采用免疫组织化学法检测FN1蛋白的阳性表达。选取胶质瘤细胞系U251和T98G，分为空白组、阴性对照组（NC）和siRNA-FN1组。采用双荧光素酶报告基因检测法研究FN1对PI3K/AKT信号通路转录活性的影响。采用MTT法检测细胞增殖，流式细胞术检测细胞周期阶段和细胞凋亡。利用β-半乳糖苷酶染色检测细胞衰老，划痕试验评估细胞迁移，Transwell试验分析细胞侵袭。分别采用蛋白质印迹法和qRT-PCR法检测收集的组织和细胞中FN1基因及PI3K/AKT通路相关基因（PI3K、AKT和PTEN）、细胞周期相关基因（pRb、CDK4和Cyclin D1）以及细胞衰老相关基因（p16和p21）的蛋白和mRNA表达水平。
GSE12657分析显示FN1是胶质瘤中上调最明显的基因。在GSE12657和GSE15824数据集中，胶质瘤组织中FN1基因表达高于正常组织。GO富集分析和KEGG通路富集分析表明，FN1参与细胞外基质（ECM）成分的合成及PI3K/AKT信号通路。研究证实了FN1基因在PI3K/AKT信号通路调控中的作用，发现沉默FN1基因可通过下调FN1基因表达及破坏PI3K-AKT信号通路抑制细胞增殖、促进细胞凋亡和衰老，并减少迁移和侵袭。
本研究结果表明，FN1通过激活PI3K/AKT信号通路在促进胶质瘤生长、侵袭和存活中发挥重要作用。
