BRD9 alteration in various types of gastric cancer
TCGA data showed that BRD9 is changed to some extent in various types of gastric cancer. Figure 1A shows that BRD9 exhibits multiple alterations, such as low mRNA, high mRNA, deep deletion, and amplification in mucinous stomach adenocarcinoma; low mRNA, high mRNA, and mutation in tubular stomach adenocarcinoma; and low mRNA, high mRNA, and deep deletion in diffuse-type stomach adenocarcinoma. Through the data processing module of analyzing cancer copy number variation, GISTIC analysis revealed that BRD9 shows deep deletion, shallow deletion, diploid, gain, amplification, and other changes in gastric cancer (Fig. 1B). TCGA database concluded that BRD9 expression is changed in 143 (30%) of 478 patients with gastric cancer (Fig. 1C). On basis of these analysis, the BRD9 alteration data of tumor cells in TCGA were employed to screen BRD9 high expression (MGC-803 cells) and normal expression (AGS cells) gastric cancer cells as cell models to discuss the influence of BRD9 on gastric cancer.
RNA-seq data and identification of DEGs in gastric cancer cells
RNA-seq was used to determine gene alteration in MGC-803 cells and AGS cells after the addition of BRD9 inhibitors, namely, BI9564 and BI7273 and to identify the DEGs in gastric cancer cells. Information on the groups is as follows:
Group MGC-803: MGC-803 cells (control group), MGC-803 cells added with BI9564 (experimental group 1, T1), and MGC-803 cells added with BI7273 (experimental group 2, T2).
Group AGS: AGS cells (control group), AGS cells added with BI9564 (experimental group 1, T1), and AGS cells added with BI7273 (experimental group 2, T2).
Results for group MGC-803: 1204 DEGs were obtained when group MGC-803 T1 dataset was screened by the limma package (corrected P-value < 0.05, logFC > 1.5). Among these DEGs, 564 were upregulated, and 640 were downregulated. Meanwhile, 1338 DEGs were obtained for group MGC-803 T2 dataset. Among which, 754 were upregulated, and 584 were downregulated. The number of overlap DEGs for group MGC-803 T1 dataset and group MGC-803 T2 dataset was 425, of which 248 were upregulated, and 177 were downregulated (Fig. 2A).
Results for group AGS: 974 DEGs were obtained for group AGS T1 dataset, of which 539 were upregulated, and 435 were downregulated genes. Meanwhile, 1006 DEGs were obtained for group AGS T2 dataset. Among which, 536 were upregulated, and 470 were downregulated. The number of overlap DEGs for group AGS T1 dataset and group AGS T2 dataset was 382, of which 245 were upregulated, and 137 were downregulated (Fig. 2B).
Heatmap R package was used for cluster analyses to draw a heatmap of DEG expression levels as shown in Figs. 2C and 2D. Group information is shown in horizontal axis, and vertical axis represents the genes. Owing to space limitation, the gene names on the right were selectively displayed. Figures 2C and D show significant difference in DEG expression between the experimental and control groups.
Analysis of DEGs in gastric cancer cells added with BRD9 inhibitor
The intersection of 425 DEGs in group MGC-803 and 382 DEGs in group AGS were analyzed by pheatmap R package. As shown in Fig. 3A, only 12 DEGs in the intersection. Through the comparison of these 12 genes (ARMC8, AVEN, MTVR2, MYH16, NPAS4, PCDH17, PLGLB2, RFPL3S, RNA28S5, SAMD9L, SHBG, and TMEM178B), we found that only three genes (MYH16, SAMD9L, and TMEM178B) had the same regulatory direction. Therefore, we think that for high BRD9 expression cell model-MGC-803 cells the controlled genes and pathways are quite different from those of AGS cells with normal BRD9 expression. Therefore, we can discuss the regulatory pathways of BRD9 in gastric cancer in two cases: one is gastric cancer (MGC-803 cells) with high BRD9 expression, the other is gastric cancer cells (AGS cells) with normal BRD9 expression.
GO and KEGG pathway enrichment analyses of DEGs in MGC-803 cells
Comprehensive bioinformatics analysis of RNA-seq data was performed using DAVID online analysis tool to obtain the biological annotation and regulated signal pathway of the DEGs controlled by BRD9 in MGC-803 cells. Figure 4 shows that the GO analysis of DEGs was divided into three functional groups, namely, molecular function, biological processes, and cell composition. In the molecular function group, the DEGs were mainly concentrated in secretion by cell, chloride transport, ion transport, intracellular signal transduction, and cell adhesion. In the cellular component group, the DEGs were mainly enriched in integral component of plasma membrane golgi lumen, integral component of membrane, and plasma membrane. In the molecular function group, the DEGs were mainly enriched in calcium ion binding, basic amino acid transmembrane transporter activity, and arginine binding. These GO analysis results indicate that most DEGs are substantially concentrated in cell secretion function, transport function, adhesion ability, signal transduction, and membrane formation. Meanwhile, KEGG pathway analysis indicated that the DEGs are mainly enriched in cell adhesion molecules, oxytocin signaling pathway, gastric acid secretion, calcium signaling pathway, and estrogen signaling pathway.
Analysis of DEGs controlled by BRD9 in group MGC-803 by using a PPI network
The DEG expression products in MGC-803 cells were constructed using the STRING database (http://string-db.org) to construct PPI networks with a total of 425 DEGs. The constructed data were inputted into Cytoscape software. Nodes that are isolated and with minimal connections with other nodes were deleted, and those with many connections with surrounding genes were highlighted. Visual analysis was also conducted. After the removal of isolated and partially connected nodes, a complex network of DEGs was constructed and is shown in Fig. 5. Among the 86 genes showing significant interaction, only three named PECAM1, SPP1, and GNB3 had the strongest interaction and were marked with red box. ANO1, ALPL, NOS3, VWF, GNAO1, ADRB1, ADCY1, CCL28, and GRM4 genes are the second most closely related genes and were marked with yellow triangle. PPI network indicated that these genes might play a key role in the MGC-803 cells controlled by BRD9.
GO and KEGG pathway enrichment analyses of DEGs in AGS cells
Comprehensive bioinformatics analysis of RNA-seq data was performed using DAVID online analysis tool to obtain the biological annotation and regulated signal pathway of the DEGs controlled by BRD9 in AGS cells, and the results are shown in Fig. 5. In the molecular function group, the DEGs were mainly concentrated in collagen catabolic process, receptor internalization, cell surface receptor signaling pathway, calcium ion transmembrane transport, positive gene regulation, and signal transduction. In the cellular component group, the DEGs were mainly enriched in integral component of plasma membrane, plasma membrane, extracellular region, integral component of membrane, and extracellular space, endosome. In the molecular function group, the DEGs were mainly enriched in extracellular matrix structural constituent, calcium ion binding, carboxypeptidase activity, interleukin-8 receptor activity, calcitonin receptor activity, interleukin-8 binding, and fibronectin binding. These GO analysis results indicate that most DEGs are remarkably concentrated in collagen catabolism, the functional pathway of receptor on cell surface, and the function of calcium ion transmembrane transport. Meanwhile, KEGG pathway analysis indicated that the DEGs are mainly enriched in protein digestion and absorption, vitamin digestion and absorption, and calcium signaling pathway.
Analysis of the DEGs controlled by BRD9 in group AGS by using a PPI network
The DEG expression products in AGS cells were constructed using the STRING database (http://string-db.org) to establish a PPI network with a total of 382 DEGs. The constructed data were inputted into Cytoscape software. Nodes that were isolated and had minimal connections with other nodes were deleted, and those with many connections with surrounding genes were highlighted. Visual analysis was also performed. After the removal of isolated and partially connected nodes, a complex network of DEGs was constructed and is shown in Fig. 7. Among the 70 genes with significant interaction, only two, namely, AR and GNGT2 had the strongest interaction and were marked with red hexagon. APOB, PRKG1, PRKGA, 7H, INSL3, RAMP1, CRHR2, HRH2, DRD5, CALCRL, CXCR1, CXCR2, CXCL10, CXCL11, GALR3, and SSTR5 genes are the second most closely related genes and were marked with yellow box. PPI network indicated that these genes might play a key role in the AGS cells controlled by BRD9.