Wang Mingliang, Lu Yida, Wang Huizhen, Wu Youliang, Xu Xin, Li Yongxiang
General Surgery Department, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Front Oncol. 2021 Dec 17;11:740120. doi: 10.3389/fonc.2021.740120. eCollection 2021.
The role of activating transcription factor 4 (ATF4) underlying gastric cancer (GC) remains unclear. The purpose of this study was to investigate the expression levels and biological functions of ATF4 in GC.
Expression of ATF4 was detected by quantitative PCR (qPCR), Western blotting, and immunohistochemistry. Cox regression was used for survival analysis and the construction of the nomogram. Immunofluorescence was used to identify the intracellular localization of ATF4. Knockdown and overexpression of ATF4 in GC cells followed by wound healing and Transwell assays, EdU and Calcein-AM/propidium iodide (PI) staining, and cell cycle detection were performed to examine its function . Transmission electron microscopy was performed to assess the autophagy levels upon ATF4 silencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) were used to determine gene enrichment. SPSS 22.0 software, GraphPad Prism 7.0, and R version 3.6.1 were used for statistical analysis.
ATF4 expression was upregulated in GC cells and tissues compared with corresponding normal tissues. Survival analysis suggested that a high ATF4 expression was strongly associated with worse overall survival (OS) of GC patients ( < 0.001). The nomogram and the receiver operating characteristic (ROC) curves demonstrated that ATF4 was a highly sensitive and specific prognostic marker of GC [C-index = 0.797, area under the ROC curve (AUC) of 3-year OS = 0.855, and AUC of 5-year OS = 0.863]. In addition, ATF4 knockdown inhibited the cell proliferation, migration, invasion, and cell cycle progression of GC cells , while overexpression of ATF4 exerted the opposite effects. Bioinformatics analysis showed that ATF4 could promote GC progression possibly by regulating asparagine (Asn) metabolism and autophagy pathways. Further experiments indicated that ATF4 expression was significantly positively correlated with ASNS expression. The inhibition of cell clone formation in Asn-deprived conditions was more significant in the shATF4 group. Finally, we found that ATF4 promoted autophagy through regulating the mTORC1 pathway in GC cells.
These findings suggested that ATF4 can significantly promote GC development and serve as an independent prognostic factor for GC.
激活转录因子4（ATF4）在胃癌（GC）中的作用尚不清楚。本研究旨在探讨ATF4在GC中的表达水平及生物学功能。
采用定量PCR（qPCR）、蛋白质免疫印迹法和免疫组织化学法检测ATF4的表达。采用Cox回归进行生存分析并构建列线图。采用免疫荧光法鉴定ATF4的细胞内定位。对GC细胞进行ATF4基因敲低和过表达，随后进行伤口愈合实验、Transwell实验、EdU和钙黄绿素-AM/碘化丙啶（PI）染色以及细胞周期检测，以研究其功能。采用透射电子显微镜评估ATF4沉默后的自噬水平。利用京都基因与基因组百科全书（KEGG）分析和基因集富集分析（GSEA）确定基因富集情况。使用SPSS 22.0软件、GraphPad Prism 7.0和R 3.6.1版本进行统计分析。
与相应正常组织相比，GC细胞和组织中ATF4表达上调。生存分析表明，ATF4高表达与GC患者较差的总生存期（OS）密切相关（<0.001）。列线图和受试者工作特征（ROC）曲线表明，ATF4是GC的一种高度敏感且特异的预后标志物[C指数=0.797，3年OS的ROC曲线下面积（AUC）=0.855，5年OS的AUC=0.863]。此外，ATF4基因敲低抑制了GC细胞的增殖、迁移、侵袭及细胞周期进程，而ATF4过表达则产生相反作用。生物信息学分析表明，ATF4可能通过调节天冬酰胺（Asn）代谢和自噬途径促进GC进展。进一步实验表明，ATF4表达与ASNS表达显著正相关。在缺乏Asn的条件下，shATF4组对细胞克隆形成的抑制作用更显著。最后，我们发现ATF4通过调节GC细胞中的mTORC1途径促进自噬。
这些研究结果表明，ATF4可显著促进GC发展，并可作为GC的独立预后因素。
