- Expression of BAZ2A in pan-cancer
We first analyzed the level of expression for BAZ2A in cancer and paracancerous tissues. By utilizing the TCGA database, Our research identified a markedly elevation in BAZ2A expression across multiple cancer types, this encompasses liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), and esophageal cancer (ESCA) (Figures 1A, B).
CPTAC analysis showed that BAZ2A protein expression was increased in diverse types of cancer (Figure 1C). To determine the association between BAZ2A expression levels and tumor pathological stage, we evaluated BAZ2A expression in patients using CPTAC analysis and found that upregulation of BAZ2A protein expression correlated with pathological stage of clear cell carcinoma, UCEC, ovarian cancer, and colon cancer (Supplementary Figure 1A). Together, these findings demonstrate that Elevated BAZ2A expression was observed in cancerous tissues and exhibited a correlation with pathological stage.
- BAZ2A phosphorylation in pan-cancer
Using the CPTAC dataset, we analyzed the levels of BAZ2A phosphorylation in four cancer types (LIHC, KIRC, OV, and HNSC). The S1395 site of BAZ2A showed higher phosphorylation levels in LIHC and KIRC tumor tissues in comparison to normal tissues, but lower phosphorylation levels in OV tumor tissues (Supplementary Figure 1B). S1768 phosphorylation levels were also higher in LIHC, OV, HNSC tumor tissues compared with normal tissues. Our results also revealed several sites with increased phosphorylation in HNSC tumor tissues, including S115, S494, S1768, Y1775, and S1776. These findings imply that alterations in the post-translational modifications of BAZ2A might contribute to the process of tumorigenesis.
- The association between BAZ2A expression and tumor diagnosis and prognosis
Subsequently, we examined the association between BAZ2A expression and patient prognosis across various types of cancer using the TCGA dataset. The survival analysis indicated that increased expression of BAZ2A was linked to a poor prognosis in patients with LIHC, KIRP, pheochromocytoma and paraganglioma (PCPG), and endometrial cancer (UCEC), while reduced expression of BAZ2A was linked to an adverse prognosis in KIRC (Figures 2A, B).
ROC curves were employed to assess the diagnostic utility of BAZ2A across multiple cancer types. BAZ2A showed some diagnostic accuracy for LIHC (0.846), KIRC (0.642), KIRP (0.522), PCPG (0.741), and UCEC (0.61) (Figure 2C). These results showed that BAZ2A expression influenced prognosis in several cancers and that BAZ2A gene expression may serve as a diagnostic marker in some cancers.
4.Genetic alterations are correlated with DNA methylation of the BAZ2A promoter.
We used two probes (cg12199011, cg20829193) to analyze TCGA RNAseq and Methylation450 data to detect DNA methylation levels of the BAZ2A gene promoter. The findings demonstrated that BAZ2A expression was inversely linked to promoter methylation in LIHC, KIRC, KIRP, PCPG, and UCEC (Figure 3A).
Analysis of gene mutation and copy number variation revealed various genetic variations of the BAZ2A gene in cancer, including mutations, structural variations, amplification, and deletions, among which mutation was the most frequently detected variation. Cutaneous melanoma (SKCM) exhibited the highest frequency of BAZ2A gene alteration (>10%) (Figure 3B). The sites and types of BAZ2A gene alterations are shown in Figure 3C. Variant of uncertain significance mutations were the major categories of genetic alterations;the R553C mutation was detected in three UCEC samples and one SKCM sample (Figure 3C, D).
Survival analysis indicated that patients with BAZ2A gene mutations had a poorer prognosis in terms of OS, PFS, DFS, DSS, and DSS in LIHC and KIRC than patients without BAZ2A gene mutation (Figures 3E, F). In UCEC, the OS, PFS, DSS of patients with BAZ2A gene mutations were better than those without BAZ2A gene mutation (Figure 3G).
- Association of BAZ2A gene expression and immune infiltration
We next evaluated the association between BAZ2A gene expression and the extent of immune cell infiltration across various cancer types using the TIMER platform. In most cancers, including LIHC, KIRC, KIRP, PCPG, and HNSC (including HNSC+HPV-, HNSC+HPV+), we observed a positive correlation between BAZ2A mRNA expression and the levels of Tregs and macrophage cell infiltration(Figure 4A). The scatterplots generated by the TIMER also demonstrated that BAZ2A expression exhibited a association with the level of invasion of immune cells in different tumors (Figure 4B). Taken together, BAZ2A gene expression is linked to the infiltration of immune cells in multiple cancer types.
- Enrichment analysis of proteins and genes associated with BAZ2A
Next, we conducted a screening for BAZ2A interacting proteins and genes, followed by enrichment analyses. Through the utilization of the STRING database, The amount is 31 BAZ2A-interacting proteins were identified, and the network of interactions among these proteins is visualized in Figure 5A. BAZ2A-related genes with the highest enrichment in biological process (BP), cellular component (CC), and molecular function (MF) are shown in Figure 5B. The KEGG analysis revealed a markedly enrichment of BAZ2A-related genes in the lysine degradation pathway, the TGF-β signaling pathway, and viral life cycle (Figures 5B, C). Analysis of RNAseq data from TCGA showed that BAZ2A and its related genes, KMT2D, CELF1, CCNT1, CREBBP, SMG1, and SRCAP genes, were co-expressed in LIHC, KIRC, KIRP, PCPG, and UCEC (Figure 5D).
- BAZ2A is highly expressed in LIHC and promotes the malignant behavior of LIHC cells
We selected LIHC to further explore the function of BAZ2A in cancer. We examined several hepatoma cell lines and found that that BAZ2A transcription levels were highest in LM6 cells (Figure 6A). Thus, we selected this cell line for subsequent experiments. We designed siRNAs against BAZ2A and confirmed their efficacy (Figure 6B). CCK8 and clonal formation experiments showed that BAZ2A downregulation inhibited cell proliferation and clonal formation of LM6 cells (Figure 6C, D). Transwell experiments showed that BAZ2A silencing reduced cancer cell migration and invasion (Figures 6E, F). Flow cytometry showed that downregulation of BAZ2A led to increased cell apoptosis, which was supported by western blot of Bax and other apoptosis-related proteins (Figure 6G, H). The function of BAZ2A in facilitating cancer cell migration was further validated by the detection of epithelial–mesenchymal transition (EMT)-related molecules (Figure 6I).
We collected tumor tissue samples from 80 patients with liver cancer and examined BAZ2A expression by immunohistochemistry. BAZ1A was highly expressed in liver cancer tissues relative to the adjacent cancer tissues in 30 samples (Figures 6J, K).
- Multiomics analysis of BAZ2A
To further validate the relationship between BAZ2A and cancer, we performed transcriptome and proteome sequencing in cells silenced for BAZ2A or transfected with NC siRNA as a negative control.The correlation analysis of transcriptomics and proteomics results indicated that upon the downregulation of BAZ2A expression, the numbers of proteins and genes with significant differences were associated,and the correlation between differential proteins and differential genes was good (Figures 7A, B).
The differentially expressed proteins identified by proteome sequencing were subjected to analysis using GO and KEGG. GO bubble plots indicated that most of the differential proteins enriched in BP were related to lipid metabolism processes, such as cellular lipid catabolic process and triglyceride catabolic process (Figure 7C). The differential proteins enriched in MF were related to lipoprotein receptor activity and lipoenzyme activity, such as triglyceride lipase activity and lipoprotein particle receptor activity (Figure 7D). The differential proteins enriched in CC were associated with lipid components, including the membrane part and lipoprotein particle (Figure 7E). These results are illustrated by directed acyclic plots in Supplementary Figures 2A–C.
Similar results were obtained from the combined proteome and transcriptome analysis. In GO analyses with significant enrichment in both omics, BP differential genes and differential proteins were also enriched in lipid metabolism processes, with 34 proteins and 328 genes enriched in the lipid catabolic process. CC-enriched differential proteins and differential genes were associated with lipid components, with 70 proteins and 1196 genes enriched in the intrinsic component of membrane (Figure 7F). The findings obtained were supported by transcriptome KEGG enrichment , in which the highest degree of enrichment was in the metabolic pathway (Figure 7G). These findings support a role for BAZ2A in lipid metabolism processes.
Figure 7G shows the PPI network of the 73 differential proteins; the core protein is SOX2 (Figure 7H). Abnormal expression of SOX2 was shown to be intricately linked to the occurrence, differentiation, metastasis, and poor prognosis of malignant tumors [18,19]. We performed KEGG enrichment analysis on the 73 differential proteins and observed that these DEPs were enriched in multiple signaling pathways of cancer, such as the RAS , MAPK signaling pathway, PI3K-AKT signaling pathway, and Hippo signaling pathway (Figure 7I). This suggests that BAZ2A may be involved in the cancer-related processes by regulating key proteins and molecules involved in these processes.