FGF13敲低的肺癌A549细胞株构建及转录组学分析

Construction and Transcriptome Analysis of Lung Cancer Cell Line A549 with FGF13 Knock-down

本研究的目的是探究成纤维细胞生长因子13(fibroblast growth factor13,FGF13)影响非小细胞肺癌发生发展的分子机制。通过构建干扰FGF13的真核表达载体并进行慢病毒包装,获得FGF13敲低的肺癌A549细胞株。利用转录组测序(RNA Sequencing,RNA-seq)技术检测基因表达谱并筛选差异表达基因(differentially expressed genes,DEGs),并对差异基因进行GO和KEGG富集分析,对部分DEGs的表达水平进行验证。采用STRING在线数据库筛选DEGs编码的蛋白相互作用网络(protein-protein interaction,PPI)中的关键基因,并通过UALCAN数据库对关键基因进行预后分析。与对照组细胞相比,FGF13敲低的肺癌A549细胞株共筛选出1114个DEGs(P≤0.05,差异倍数≥2),其中表达上调和下调的DEGs分别有672和442个。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)验证CXCL10和SELE等6个基因与测序结果一致。GO富集分析显示,DEGs与细胞凋亡、细胞迁移、氧化还原等生物学过程有关。KEGG富集分析显示,DEGs与p53信号通路、TNF信号通路、PI3K-AKT信号通路等有关。在DEGs编码蛋白的相互作用网络中,CXCL10、CCL5、IL6、DDX58、BST2、CSF2、CCL4、IFIH1、CMPK2和CXCL1作为FGF13调控的潜在关键基因,其中的DDX58、IFIH1等与患者的预后显著相关。本研究经转录组测序分析揭示了FGF13在肺癌A549细胞中调控的DEGs及其参与的相关信号通路和生物学过程,为后续研究FGF13在肺癌细胞中的生物学功能及其分子机制提供科学依据。

This study aims to explore the molecular mechanism of the effect of fibroblast growth factor 13 on the occurrence and development of non-small cell lung cancer.A lung cancer cell line A549 with FGF13 knock-down was obtained by constructing eukaryotic expression vector interfering with FGF13 and packaging with lentivirus.RNA Sequencing(RNA-seq)was used to detect gene expression profile and screen differentially expressed genes.The differentially expressed genes were enriched and analyzed by GO and KEGG to verify the expression level of some DEGs.The key genes in the protein-protein interaction(PPI)network encoded by DEGs were screened by STRING online database,and the prognosis of key genes was analyzed by UALCAN database.Compared with the control group,a total of 1114 DEGs were screened in lung cancer cell line A549 with FGF13 knock-down(P≤0.05,difference multiple≥2),of which 672 and 442 DEGs were up-regulated and down-regulated respectively.Six genes including CXCL10 and SELE were verified by real-time quantitative PCR(RT-qPCR),and the results were consistent with the results of sequencing.GO enrichment analysis showed that DEGs was related to biological processes such as apoptosis,cell migration and redox.KEGG enrichment analysis showed that DEGs was related to p53 signal pathway,TNF signal pathway,PI3 K-AKT signal pathway and so on.In the interaction network of DEGs-encoded proteins,CXCL10,CCL5,IL6,DDX58,BST2,CSF2,CCL4,IFIH1,CMPK2 and CXCL1 were potentially key genes regulated by FGF13,among which DDX58 and IFIH1 were significantly related to the prognosis of patients.In this study,transcriptome sequencing analysis revealed the DEGs regulated by FGF13 in lung cancer A549 cells and its related signal pathways and biological processes,which provided a scientific basis for the follow up study of the biological function and molecular mechanism of FGF13 in lung cancer cells.