基于数据库分析FOXG1在非小细胞肺癌中的表达和临床意义及稳定过表达FOXG1肺癌细胞株A549的构建

ANALYSIS OF THE EXPRESSION AND CLINICAL SIGNIFICANCE OF FOXG1 IN NON-SMALL CELL LUNG CANCER BASED ON THE DATABASE AND THE CONSTRUCTION OF A LUNG CANCER CELL LINE A549 WITH STABLE OVEREXPRESSION OF FOXG1

通过TCGA数据库分析FOXG1在非小细胞肺癌中的表达及预后相关性,建立稳定过表达人源FOXG1基因的肺癌细胞株A549。利用TCGA数据库中下载基因表达数据和临床信息,分析FOXG1在非小细胞肺癌和正常组织的表达差异、FOXG1表达水平与临床病理特征及生存预后的相关性并进行基因集富集分析。通过HEK-293T包装慢病毒表达载体,收集病毒上清液侵染A549细胞,嘌呤霉素筛选稳定过表达FOXG1的A549细胞株。细胞核染色鉴定外源FOXG1表达定位,Western blot检测外源FOXG1的表达情况。结果发现FOXG1在非小细胞肺癌组织中高表达,且FOXG1高表达能够降低患者总体生存率。FOXG1的表达水平与患者年龄相关,与性别,分级以及TMN分期无关;细胞周期、P53、Notch等信号通路在高表达FOXG1的非小细胞肺癌组织中被激活;慢病毒表达载体共转染HEK-293T细胞成功;病毒上清液侵染A549细胞,24 h后可见绿色荧光表达,72 h后对照组空载体病毒颗粒侵染效率高达80%左右,实验组过表达FOXG1病毒颗粒侵染效率为50%~60%;经嘌呤霉素筛选培养后,对照组和实验组荧光效率均达到90%以上;细胞核染色外源FOXG1基因定位在细胞核中,Western blot结果显示外源FOXG1在细胞中正确表达。因此可以认为FOXG1基因在非小细胞肺癌患者中高表达,其表达水平与患者总体预后有关且稳定表达FOXG1的A549肺癌细胞株构建成功。

In this paper,based on the TCGA database,the expression and prognosis correlation of FOXG1 in non-small cell lung cancer were analyzed,and a lung cancer cell line A549 with stably overexpressed human FOXG1 gene was established.We downloaded the gene expression data and clinical information from the TCGA database,analyzed the expression difference of FOXG1 in non-small cell lung cancer and normal tissues,the correlation of FOXG1 expression level with clinicopathological characteristics and survival prognosis,and performed gene set enrichment analysis.The lentiviral expression vector was packaged by HEK-293T,and the virus supernatant was collected to infect A549 cell line,and puromycin screening was performed to establish an A549 cell line stably overexpressing FOXG1.The expression and localization of exogenous FOXG1 were identified by nuclear staining,and the expression of exogenous FOXG1 was detected by Western blot.The results showed that FOXG1 was highly expressed in non-small cell lung cancer tissues,and high expression of FOXG1 could reduce the overall survival rate of patients.The expression level of FOXG1 was related to the age of patients,but not related to gender,grade and TMN stage;cell cycle,P53,Notch and other signaling pathways were activated in non-small cell lung cancer tissues with high expression of FOXG1;Lentiviral expression vector was successfully co-transfected into HEK-293T cells.the virus supernatant infected A549 cell line,green fluorescence expression was visible after 24 h,and the infection efficiency of the empty vector virus particles in the control group was as high as about 80%after 72 h.The infection efficiency of the experimental group overexpressing FOXG1 virus particles was 50%-60%.After being filtered by puromycin,the fluorescence efficiency of both the control groups and the experimental groups reached more than 90%.The staining experiment showed that the exogenous FOXG1 gene was located in the nucleus,Western blot showed that exogenous FOXG1 was correctly expressed.Therefore,it can be considered that FOXG1 gene is highly expressed in patients with non-small cell lung cancer,and its expression level is related to the overall prognosis of patients.The A549 cell line stably expressing FOXG1 was successfully constructed.