高表达CRTAC1通过调控PI3K信号通路促进胃癌细胞增殖、迁移及免疫浸润

doi:10.12122/j.issn.1673-4254.2024.12.19

摘要/Abstract

摘要：

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。

关键词: CRTAC1, 胃癌, 免疫浸润, 上皮间质转化

Abstract:

Objective 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. Conclusion 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.

Key words: gastric cancer, CRTAC1, immune infiltration, epithelial-mesenchymal transition

张富星, 刘国庆, 董锐, 高磊, 陆伟晨, 高连霞, 赵忠扩, 陆飞, 刘牧林. 高表达CRTAC1通过调控PI3K信号通路促进胃癌细胞增殖、迁移及免疫浸润[J]. 南方医科大学学报, 2024, 44(12): 2421-2433.

Fuxing ZHANG, Guoqing LIU, Rui DONG, Lei GAO, Weichen LU, Lianxia GAO, Zhongkuo ZHAO, Fei LU, Mulin LIU. High expression of CRTAC1 promotes proliferation, migration and immune cell infiltration of gastric cancer by regulating the PI3K/AKT signaling pathway[J]. Journal of Southern Medical University, 2024, 44(12): 2421-2433.

图/表 7

图1 CRTAC1在胃癌中表达水平与患者的预后相关

Fig.1 CRTAC1 expression level in gastric cancer is significantly correlated with the patients' prognosis. A: Pan-cancer analysis of TIMER2.0 database. B: Mutation location of CRTAC1 in gastric cancer transcriptome data of cBioPortal database. C, D: Differential analysis of CRTAC1 expression in unpaired and paired gastric cancer transcriptome data of TCGA database. E-H: Differential analysis of CRTAC1 expression in transcriptome data of BEST website of GSE18105, GSE21510, GSE39582 and GSE41258 transcriptome data in BEST website. I: Differentially expressed genes between high and low CRTAC1 expression groups in TCGA database gastric cancer transcriptome data and plotting of volcano plots. J: Expression level of CRTAC1 in TCGA database gastric cancer transcriptome data for ROC curves plotting. K: Relationship between CRTAC1 expression level and gastric cancer patients' overall survival (OS). L, M: One-way Cox and multifactorial Cox analysis of CRTAC1 in gastric cancer patients. *P<0.05,**P<0. 01, ***P<0. 001.

图2 CRTAC1表达影响胃癌肿瘤微环境

Fig.2 CRTAC1 expression affects tumor microenvironment in gastric cancer. A: Stromal cell scores, immune cell scores and tumor microenvironment scores of patients in the high and low CRTAC1 expression groups. B, C: Differential and correlation analyses of immune cells in patients in high and low CRTAC1 expression groups. D: Correlation analysis of immune checkpoints in patients in high and low CRTAC1 expression groups. *P<0.05, **P<0.01, ***P<0.001.

图3 CRTAC1在胃癌中的表达水平影响肿瘤突变负荷及预后

Fig.3 CRTAC1 expression level in gastric cancer affects tumor mutation load and prognosis. A: Scatter plot of correlation between CRTAC1 expression and TMB. B: Waterfall plot of the top 20 genes with mutation frequency in samples from high and low CRTAC1 expression groups. C: Kaplan-Meier curve of TMB affecting the overall survival of gastric cancer patients. D: Box line plot of TMB combined affecting the risk scores of gastric cancer patients. E: Kaplan-Meier curves of TMB affecting the risk scores of gastric cancer patients' overall survival. F: Kaplan-Meier curves of the effect of TMB combined with CRTAC1 expression on overall survival of gastric cancer patients. *P<0.05, **P<0. 01, ***P<0. 001.

图4 CRTAC1高表达促进胃癌细胞的生长增殖

Fig. 4 High expression of CRTAC1 promotes proliferation of gastric cancer cells. A, B: Detection of proliferative capacity of the cells with CRTAC1 knockdown using CCK8 assay. C-F: Colony formation assay of the cells with CRTAC1 knockdown. G-J: EdU assay for assessing proliferation of the cells with CRTAC1 knockdown (Original magnification: ×20). K-N: Flow cytometry for assessing cell cycle changes in the cells with CRTAC1 knockdown. **P<0. 01, ***P<0. 001 vs CRTAC1 SiNC group.

图5 CRTAC1的高表达抑制胃癌细胞凋亡

Fig.5 High expression of CRTAC1 significantly inhibits apoptosis in gastric cancer cells. A-H: Flow apoptosis assay and Hoechst assay showing that CRTAC1 knockdown promotes apoptosis of gastric cancer cells. I-J: Spearman's statistical analysis of the changes in BAX and BCL-2 expressions following CRTAC1 knockdown using the R language (×20). K-M: Changes in BAX and BCL-2 expressions in GC cells with CRTAC1 knockdown detected by Western blotting. *P<0.05, **P<0. 01, ***P<0. 001 vs CRTAC1 SiNC group.

图6 CRTAC1高表达促进胃癌细胞的增殖迁移

Fig.6 High expression of CRTAC1 promotes proliferation and migration of gastric cancer cells. A-H: Scratch wound healing assay and Transwell assay for assessing migration ability of the cells with CRTAC1 knockdown (×20). I-J: Spearman's statistical analysis of the correlation between E-cadherin (CDH1) and Vimentin (VIM) expression using R language. K-M: Western blotting of E-cadherin and vimentin protein expressions in the cells with CRTAC1 knockdown. **P<0. 01, ***P<0. 001 vs CRTAC1 SiNC group.

图7 CRTAC1通过PI3K信号通路促进胃癌细胞增殖及迁移

Fig. 7 High expression of CRTAC1 promotes proliferation and migration of gastric cancer cells through the PI3K signaling pathway. A: Circle plot of co-expression analysis of CRTAC1 performed in gastric cancer genome (red, white, and green scatters are positively, non-significantly, and negatively correlated genes, respectively, with CRTAC1 expression). B: Heat map of differential genes between high and low CRTAC1 expression groups in gastric cancer (red, gray, and blue scatters are positively, non-significantly, and negatively correlated genes, respectively, with CRTAC1 expression). C, D: Histograms of GO and KEGG enrichment analysis of CRTAC1 differential genes, FDR<0.05. E, F: Spearman's statistical analysis of the correlation between AKT2 expressions using R language. G-I: Western blotting of the PI3K signaling pathway protein expressions in cells with CRTAC1 knockdown. *P<0.05, **P<0. 01, ***P<0. 001 vs CRTAC1 SiNC group.

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