MFAP2 enhances cisplatin resistance in gastric cancer cells by regulating autophagy

Bioinformatics analysis

The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was employed to acquire the matrices about GC. The several screening criteria were set as follows: (1) samples with normal controls and GC patients; (2) the number of samples ≥10. Finally, 4 expression matrices were selected for this study, namely GSE54129, GSE65801 (Li et al., 2015), GSE79973 (Jin et al., 2017) and GSE103236 (Necula et al., 2020). By applying The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/), we downloaded the stomach gene expression data (TCGA-STAD) and clinical data (Shao et al., 2021).

When conducting the DEGs analysis, we firstly conversed the ID from the GEO database based on the platform information corresponding to the chip, and then the limma package was used for correction (Ji et al., 2021). The data were grouped into two categories (normal vs tumor), and the DEGs were further identified with the standard of —Fold Change—>2, P <0.05. For next generation sequencing data of the TCGA database, one gene annotation GTF file was used for ID transformation, and edgeR package was applied to analyze the normal and tumor groups. The DEGs in the TCGA database were screened out based on the same standard as above. The up- and down-regulated genes of the four chips in the GEO database were intersected respectively, and we took the intersection of DEGs in the TCGA and GEO databases. The intersecting DEGs were the final differential genes of GC.

R package was applied for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the above DEGs (Zhou, Lu & Xiong, 2021). Differential expression boxplots of the DEGs were drawn to verify the correctness of differential expression. Furthermore, survival analysis was carried out according to the survival information from the TCGA database, and Kaplan–Meier curves were drawn (Wang et al., 2021). For genes with significant difference in survival, clinical correlation analysis was further conducted according to the clinical data from the TCGA database. The diagnostic value of the genes was analyzed by a ROC curve, and the area under the curve (AUC) of 3 and 5 years were calculated, respectively (Cheng et al., 2019).

Cell culture

Procell Life Science&Technology Co., Ltd. (Wuhan, China) sold us human GC cell line AGS. CDDP was purchased from Qilu Pharmaceutical Co., Ltd. (Jinan, China) and dissolved with normal saline. Autophagy inducer rapamycin (Rap) was acquired from Selleck (Shanghai, China). The AGS was selected as drug-sensitive cell line. AGS/CDDP cell line was developed by gradually increasing the drug concentration (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 µg/mL CDDP, respectively) over a period of 12 months. Before the experiment, the AGS/CDDP were cultured in CDDP-free culture medium for a duration of two weeks (Peng et al., 2020).

MTT assay

AGS or AGS/CDDP was seeded into 96-well plates for 24 h at 1 ×10³ cells per well, and then received various treatments for 48 h. The medium including 0.5 mg/ml MTT reagent was put into the 96-well plates for 4 h. Finally, DMSO was applied to make formazan completely dissolved. The absorbance at 490 nm was recorded by Synergy Neo2 Hybrid Multi-Mode Reader (BioTek, El Segundo, CA, USA) (Lin et al., 2021).

Western blot

Total protein from the experimental cells was extracted with RIPA lysis buffer and quantified by bicinchoninic acid (BCA) assay. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to separate the proteins, and then the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., Burlington, MA, USA) (Wang et al., 2019). Then the membrane was blocked with 5% skim milk and incubated with the primary antibody MFAP2 (A10230, 1:2000; ABclone, Wuhan, China), β-actin (ab8227, 1:10000; Abcam, Cambridge, UK), LC3II (ab192890, 1:2000; Abcam), cleaved-caspase-3 (ab32042, 1:500; Abcam), cleaved-PARP (ab32064, 1:1000; Abcam), cleaved-caspase-9 (ab32539, 1:1000; Abcam), Beclin 1 (ab207612, 1:1000; Abcam), ATG5 (ab108327, 1:1000; Abcam) and P62 (ab56416, 1:1000; Abcam) at 4 °C overnight and the second antibody (ab7090 or ab47827; Abcam), respectively. Finally, ChemiDoc XRS+ System (Bio-rad, Hercules, CA, USA) was applied to capture chemiluminescence photos.

Apoptosis assay

The apoptosis analysis was performed through Annexin V-fluorescein isothiocyanate (FITC) / propidium iodide (Annexin V-FITC/PI) staining (Zheng et al., 2017). MultiSciences sold us Annexin V-FITC/PI apoptosis kit (catalog number: 70-AP101-100). AGS or AGS/CDDP was seeded into 6-well plates, and dissociated into single cells by EDTA-free trypsin digestion. After being washed, the cells were re-suspended in 400 µl binding buffer and incubated with 5 µl annexin V-FITC and 10 µl PI for 20 min in the dark. After filtration, utilizing flow cytometer (CytoFLEX; Beckman Coulter, Brea, CA, USA), the apoptosis rate was determined with CytExpert software.

Cell transfection

AGS or AGS/CDDP was seeded into 6-well plates at 1 ×10⁵ cell/mL and grown to the confluence of 80%. Then, the MFAP2 over-expression plasmid (OE-MFAP2, 4 μg) or MFAP2 siRNA (siMFAP2, 100 pmol) was transfected employing Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) under the guidance of standard protocol. Finally, cells were incubated overnight.

Statistical analysis

The experimental data were presented graphically utilizing GraphPad Prism 6.0 software and statistically analyzed using SPSS software (version 16.0). All experiments in this study were repeated 3 times independently. Data were indicated as mean ± SD. Comparisons were made between groups employing the Student’s t-test, and the analysis of variance (ANOVA) was applied among multiple groups. ^∗P <0.05, ^∗∗P <0.01 and ^∗∗∗P <0.001 were statistically significant.

Database analysis

The GPL type and case information of the GEO datasets were shown in Table 1. There are 235 samples (163 GC samples and 72 non-GC samples), and they are mainly Chinese samples. The clinical characteristics from the TCGA dataset were list in Table 2, including 378 GC cases.

Differential expression analysis

First, the limma package was applied to make the four chips normalized (Fig. 1A). The right graphs were normalized. Then, the DEGs were screened out in GC and non-GC groups according to the above standard. Volcano maps (Fig. 1B) and heat maps (Fig. 1C) were drawn for the results of the four chips. The intersection of up- or down-regulated genes obtained from the GEO database was taken respectively, and there were 10 up-regulated genes (BGN, INHBA, CTHRC1, SULF1, THBS2, FAP, MFAP2, SFRP4, SPP1, and COL10A1) and three down-regulated genes (MAL, GIF, and ATP4B) (Fig. 1D). Based on TCGA-STAD, the DEGs were screened out by edgeR package, and the volcano map (Fig. 1E) and heat map (Fig. 1F) were plotted. Finally, the intersection of DEGs in the TCGA database and the GEO database was taken, and a total of 10 up-regulated genes (BGN, INHBA, CTHRC1, SULF1, THBS2, FAP, MFAP2, SFRP4, SPP1, and COL10A1) and two down-regulated genes (MAL and ATP4B) were acquired (Fig. 1G).

Functional pathway enrichment analysis

The above 10 up-regulated genes and two down-regulated genes were used to establish a protein-protein interaction (PPI) network (Fig. 2A). GO enrichment analysis (Fig. 2B, Table 3) and KEGG enrichment analysis (Fig. 2C) were performed (P <0.05 and P.adjust <0.05). These genes were enriched in the GO pathways of extracellular matrix organization, extracellular structure organization, collagen-containing extracellular matrix, extracellular matrix structural constituent, and wnt-protein binding. KEGG enrichment analysis showed that THBS2 and SPP1 were enriched in the pathway of extracellular matrix (ECM)-receptor interaction.

Boxplot analysis of gene expression

The expression analysis of the 12 genes was performed. The boxplots were plotted in different datasets: GSE54129 (Fig. 3A), GSE65801 (Fig. 3B), GSE79973 (Fig. 3C), GSE103236 (Fig. 3D) and TCGA (Fig. 3E). It showed that ATP4B and MAL were downregulated in GC, while BGN, COL10A1, CTHRC1, FAP, INHBA, MFAP2, SFRP4, SPP1, SULF1 and THBS2 were upregulated in GC.

Survival analysis

To determine the influence of the above 12 genes on prognosis, survival analysis was performed based on the TCGA data (Fig. 4). According to the results, FAP, INHBA and MFAP2 had significant effects on survival (P = 0.045, 0.037, 0.034), so they were used for subsequent analysis.

Clinical correlation analysis

FAP, INHBA and MFAP2 were subjected to clinical correlation analysis according to the TCGA clinical data (Fig. 5). The clinical information of samples was shown in Table 2. According to the correlation analysis results, FAP was correlated with Grade, Stage and T stage, and both INHBA and MFAP2 were related to T stage.

ROC analysis

ROC analysis was conducted on the above three genes based on the survival time and survival status from the TCGA. The ROC-curves of 3-year and 5-year survival prediction were plotted respectively. According to the outcomes, the accuracy of 5-year survival prediction was more than 0.75, and FAP (Fig. 6A), INHBA (Fig. 6B) and MFAP2 (Fig. 6C) can be good diagnostic factors of GC. Among the three genes, the AUC of MFAP2 in the ROC-curves of 3-year and 5-year survival prediction were all more than 0.7. In this study, MFAP2 was selected for subsequent research.

MFAP2 was upregulated in AGS/CDDP cells

AGS/CDDP was established by concentration gradient induction. 3 µg/ml CDDP was supplied into the medium of AGS and AGS/CDDP cells. MTT assay showed that the cell viability of AGS/CDDP was significantly higher than AGS (Fig. 7A). Annexin V-FITC/PI staining suggested that the cell apoptosis rate of AGS/CDDP was significantly reduced (Fig. 7B). Pro-apoptotic protein cleaved PARP, cleaved caspase-3, and cleaved caspase-9 were downregulated in AGS/CDDP (Fig. 7C). The results of MTT and cell apoptosis analysis showed that drug-resistant cell line AGS/CDDP was obtained. Furthermore, western blot suggested that MFAP2 was upregulated in AGS/CDDP cells in contrast with AGS cells (Fig. 7D).

Effect of MFAP2 on CDDP sensitivity of AGS

To further explore the action of MFAP2 on CDDP sensitivity, we transfected MFAP2 overexpressed plasmid (OE-MFAP2) and siMFAP2 in AGS and AGS/CDDP cells, respectively. Western blot results showed that MFAP2 was upregulated in the AGS+OE-MFAP2 in contrast with the AGS, and apparently downregulated in the AGS/CDDP+siMFAP2 compared to the AGS/CDDP (Fig. 8A). In comparison with the AGS, the IC50 was increased (17.79 vs 3.04 µg/mL, Fig. 8B), the cell apoptosis was reduced (Fig. 8C), and the sensitivity to CDDP were decreased in the AGS+OE-MFAP2 group. Additionally, in contrast with the AGS/CDDP, the AGS/CDDP+siMFAP2 group showed a lower IC50 (5.47 vs 18.21 µg/mL, Fig. 8B), a higher level of cell apoptosis (Fig. 8C), and an increased sensitivity to CDDP. Western blot was applied to detect the expression of pro-apoptotic protein, the result of which was consistent with that of Annexin V-FITC/PI staining (Fig. 8D). In this section, we confirmed that MFAP2 can regulate the CDDP sensitivity of AGS.

Effect of MFAP2 on the autophagy in AGS

To explore the mechanism of MFAP2 regulating CDDP sensitivity, the effect of MFAP2 on cell autophagy was further studied. After cells were cultured at 3 µg/mL CDDP for 48 h, the expression of autophagy-related proteins was determined (Fig. 9). The results of western blot displayed that compared with the AGS group, p62 in the AGS+OE-MFAP2 group was downregulated, while Beclin-1, ATG5 and LC3 II were upregulated, and autophagy was enhanced. Compared to the AGS/CDDP, the expression of p62 in the AGS/CDDP+siMFAP2 was upregulated, while Beclin-1, ATG5 and LC3 II expression were downregulated, suggesting that autophagy was decreased. We preliminarily confirmed that MFAP2 plays an important role on autophagy on the basis of these results.

MFAP2 enhanced CDDP resistance by promoting autophagy

To further demonstrate the enhancement of MFAP2 on CDDP resistance by promoting autophagy, the cells were grouped as follows: AGS, AGS/CDDP, AGS/CDDP+siMFAP2, and AGS/CDDP+siMFAP2+Rap (100 nM, 24h). Western blot results suggested that the protein content of MFAP2 was the highest in the AGS/CDDP, and MFAP2 was significantly downregulated in the AGS/CDDP+siMFAP2 and AGS/CDDP+siMFAP2+Rap groups after the transfection with siMFAP2 (Fig. 10A). Compared with the AGS group, the level of autophagy (Fig. 10B) and the CDDP resistance (Fig. 10C–E) were increased in the AGS/CDDP group. After MFAP2 expression was down-regulated, autophagy in the AGS/CDDP+siMFAP2 was weakened (Fig. 10B), and the sensitivity to CDDP was enhanced (Figs. 10C–10E). Further, autophagy was enhanced in the AGS/CDDP+siMFAP2+Rap group (Fig. 10B), and the resistance to CDDP was increased (Figs. 10C–10E) compared with the AGS/CDDP+siMFAP2 group. Overall, we further identified that MFAP2 enhanced CDDP resistance by promoting autophagy.