MFAP2 enhances cisplatin resistance in gastric cancer cells by regulating autophagy

Abstract

Background: Cisplatin (CDDP) is of importance in cancer treatment and widely used in advanced gastric cancer (GC). However, its clinical usage is limited due to its resistance, and the regulatory mechanism of CDDP resistance in GC has not yet been fully elucidated. In this study, we first conducted a comprehensive study to investigate the role of MFAP2 through bioinformatics analysis.

Methods: The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were applied to downloadgene expression data and clinicopathologic data, and the differentially expressed genes (DEGs) were further analyzed. Then, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and survival analysis were conducted. Furthermore, according to the clinicopathological characteristics of TCGA, clinical correlation analysis was conducted, and a receiver operating characteristic curve (ROC) was plotted.

Results: We revealed that FAP, INHBA and MFAP2 were good diagnostic factors of GC. However, the mechanism of MFAP2 in GC remains elusive, especially in the aspect of chemotherapy resistance. We developed the CDDP-resistant cell line, and found that MFAP2 was upregulated in CDDP-resistant cells, and MFAP2-knockdown improved CDDP sensitivity. Finally, we found that MFAP2 enhanced CDDP resistance by inducing autophagy in drug-resistant cell lines.

Conclusions: The above results suggested that MFAP2 could affect the chemotherapy resistance by altering the level of autophagy in GC patients as a potential therapeutic target.

Keywords: Autophagy; CDDP-resistance; Gastric cancer; MFAP2.

Figure 1. Differential expression analysis.

(A) Data normalization; (B) volcano map of DEGs (four GEO datasets); (C) heat map of DEGs (four GEO datasets); (D) intersection of DEGs obtained from four GEO datasets; (E) volcano map of DEGs (TCGA database); (F) heat map of DEGs (TCGA database); (G) the intersection of DEGs based on the TCGA database and the GEO database.

Figure 2. Functional pathways analysis.

(A) PPI network construction; (B) GO analysis; (C) KEGG analysis.

Figure 3. Boxplot analysis of differential expression.

(A) GSE54129; (B) GSE65801; (C) GSE79973; (D) GSE103236; (E) TCGA database.

Figure 4. Survival analysis of DEGs.

Figure 5. Clinical correlation analysis of FAP, INHBA and MFAP2.

Figure 6. ROC analysis of FAP, INHBA and MFAP2.

(A) ROC analysis of FAP; (B) ROC analysis of INHBA; (C) ROC analysis of MFAP2.

Figure 7. Cell differences between AGS and AGS/CDDP.

(A) The cell viability of AGS and AGS/CDDP detected by MTT (P < 0.0001); (B) Apoptosis rate measured by Annexin V-FITC/PI assay (P < 0.0001); (C) cleaved PARP, cleaved caspase-9, and cleaved caspase-3 displayed by western blot (P = 0.0055, 0.0011, 0.0018); (D) MFAP2 detected by western blot (P = 0.0023).

Figure 8. Effect of MFAP2 on CDDP sensitivity of AGS.

(A) The expression of MFAP2 (compared with AGS, P = 0.0113; compared with AGS/CDDP, P = 0.0136); (B) the cell viability at different concentrations of CDDP (IC50); (C) cells were cultured at 3 µg/mL CDDP for 24 h, and Annexin V-FITC/PI staining assay was applied to observe apoptosis rate (compared with AGS, P = 0.0375; compared with AGS/CDDP, P = 0.0098); (D) cells were cultured at 3 µg/mL CDDP for 48 h, and the content of pro-apoptotic proteins (cleaved PARP, cleaved caspase-3, and cleaved caspase-9) was detected (compared with AGS, P = 0.0009, 0.0025, 0.0089; compared with AGS/CDDP, P = 0.0023, 0.035, 0.0003).

Figure 9. The expression of p62, Beclin-1, ATG5 and LC3 I/II.

Compared with AGS, P = 0.016, 0. 0011, 0.0023, 0.016; compared with AGS/CDDP, P = 0.0034, 0.0092, 0.0005, 0.0106.

Figure 10. MFAP2 promoted autophagy and enhanced CDDP resistance of AGS.

(A) The expression of MFAP2 (compared with AGS, P = 0.0047; compared with AGS/CDDP, P = 0.0036); (B) the protein content of p62, Beclin-1, ATG5 and LC3 I/II (compared with AGS, P = 0.0001, 0.00002, 0.0005, 0.00004; compared with AGS/CDDP, P = 0.011, 0.007, 0.0442, 0.011; compared with AGS/CDDP+siMFAP2, P = 0.0021, 0.0003, 0.0023, 0.0011); (C) the cell viability at different concentrations of CDDP (IC50); (D) the cell apoptosis after treatment with 3 µg/ml CDDP (compared with AGS, P = 0.0003; compared to AGS/CDDP, P = 0.0005; compared to AGS/CDDP+siMFAP2, P = 0.0227); (E) the protein content of cleaved PARP, cleaved caspase-3, and cleaved caspase-9 in the cells treated with 3 µg/ml CDDP (compared with AGS, P < 0.0001; compared to AGS/CDDP, P = 0.0002, 0.0001, 0.0031; compared with AGS/CDDP+siMFAP2, P = 0.0081, 0.012, 0.029).