High expression of CRTAC1 promotes proliferation, migration and immune cell infiltration of gastric cancer by regulating the PI3K/AKT signaling pathway

doi:10.12122/j.issn.1673-4254.2024.12.19

Abstract

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

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.

Figures/Tables 7

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.

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.

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.

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.

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.

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.

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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