Objectives: To investigate the expression of cartilage acidic protein 1 (CRTAC1) in gastric cancer (GC) and its effect on biological behaviors and immune cell infiltration of GC.
Methods: Transcriptomic, GO and KEGG analyses were conducted to investigate the association of CRTAC1 expression with prognosis of GC patients and its involvement in cell function and signaling pathways. ESTIMATE algorithm was used to analyze the effect of CRTAC1 expression on the tumor microenvironment and the tumor mutation load. In two GC cell clines (HGC-27 and MKN-74), CCK8, EdU and clone formation assays, flow cytometry, and Hoechst staining were used to examine the effects of CRTAC1 knockdown on cell proliferation, cell cycle changes and apoptosis. Wound healing assay, Transwell assay, and Western blotting were performed to analyze the effect of CRTAC1 knockdown on GC cell migration and the underlying mechanism.
Results: Bioinformatics analysis showed significantly higher expression of CRTAC1 in GC tissues than in adjacent tissues (P<0.05). Age and tumor stage were both prognostic risk factors in GC patients with high CRTAC1 expression (P<0.001). Analysis using ESTIMATE algorithm showed that CRTAC1 expression increased immune cell infiltration and decreased tumor mutational load in GC (P<0.001). In HGC-27 and MKN-74 cells, CRTAC1 knockdown significantly inhibited cell proliferation and migration and promoted cell apoptosis. Western blotting demonstrated that CRTAC1 knockdown significantly increased E-cadherin expression and reduced the expression levels of vimentin, p-PI3K, AKT2, p-AKT and p-mTOR in GC cells.
Conclusions: High expression of CRTAC1 in GC tissues affects immunotherapeutic efficacy and prognosis of the patients, possibly by promoting epithelial-mesenchymal transition via modulating tumor mutational load, tumor microenvironment, and the PI3K/AKT signaling pathway.
目的: 探究软骨酸性蛋白1（CRTAC1）参与影响胃癌生物学行为和免疫浸润的机制。方法: 采用转录组分析CRTAC1在胃癌肿瘤细胞中表达与预后的关系，GO和KEGG富集分析CRTAC1表达水平可能参与细胞的功能及信号通路。ESTIMATE算法分析CRTAC1表达对肿瘤微环境及肿瘤突变负荷的影响。CCK-8、EdU、克隆形成以及流式细胞周期实验检测CRTAC1参与胃癌细胞的增殖。流式细胞凋亡、Hoechst和Western blotting实验检测CRTAC1抑制胃癌细胞凋亡。划痕伤口愈合、Transwell以及Western blotting实验分析CRTAC1参与胃癌迁移的机制。结果: 生物信息学分析结果显示：CRTAC1在胃癌和癌旁组织中的表达量差异有统计学意义（P<0.05）；单因素Cox和多因素Cox回归分析表明年龄和分期分别是CRTAC1高表达胃癌患者的预后风险因素（P<0.001）。采用ESTIMATE算法结果显示：CRTAC1的表达增加免疫细胞浸润丰度（P<0.001）；同时CRTAC1高表达减少肿瘤突变负荷（P<0.001）。采用CCK-8、EdU、克隆形成实验检测敲低CRTAC1后明显抑制细胞增值（P<0.001）；流式细胞周期实验检测敲低CRTAC1后细胞阻滞在G1期（P<0.001）。流式细胞凋亡、Hoechst实验结果显示敲低CRTAC1后细胞凋亡增加（P<0.001）；生物信息学预测胃癌患者中CRTAC1与凋亡相关蛋白BAX和BCL2的关系（P<0.05），Western blotting实验验证敲低CRTAC1后CRTAC1、BAX和BCL2的表达水平变化（P<0.05）。划痕伤口愈合、Transwell实验显示，敲低CRTAC1可抑制细胞的增值迁移（P<0.001）；生物信息学预测胃癌患者中CRTAC1与EMT相关蛋白E-cadherin和Vimentin与CRTAC1的关系（P<0.05），Western blotting实验验证敲低CRTAC1后E-cadherin和Vimentin的表达水平改变（P<0.01）。KEGG富集分析显示，CRTAC1生物功能可能与PI3K/AKT信号相关，Western blotting实验证实CRTAC1可促进p-PI3K、AKT2、p-AKT、p-mTOR的表达（P<0.05）。结论: CRTAC1在胃癌组织中高表达影响患者的免疫治疗效果和预后，可能通过调控肿瘤突变负荷和肿瘤微环境，以及PI3K/AKT信号通路促进胃癌细胞EMT进程相关。.
Keywords: CRTAC1; epithelial-mesenchymal transition; gastric cancer; immune infiltration.