3.1 Expression of CHAF1B mRNA was up-regulated in LUAD

First, the expression levels of CHAF1B mRNA in various malignant tumor types were determined using the GEPIA2 database. These findings indicated that CHAF1B mRNA was up-regulated in BLCA, BRCA, CESC, COAD, DLBC, LUAD, LUSC, READ, SKCM, STAD and THYM, but downregulated in LAML cells (Fig. 1A, B). According to the UALCAN database, CHAF1B mRNA expression level in LUAD tissues was considerably higher than that in normal tissues (Fig. 1C). Additionally, patients with TP53 mutations exhibited high CHAF1B mRNA expression levels (Fig. 1D). According to the ROC curve, the AUC of CHAF1B mRNA in LUAD was 0.963, suggesting its potential as a diagnostic tool for early detection (Fig. 1E). KM analysis indicated that patients with high levels of CHAF1B mRNA expression exhibited short survival times [hazard ratio (HR) = 1.5, p = 0.0098] (Fig. 1F). Consequently, CHAF1B may influence the prognosis of LUAD and could be used to develop novel biomarkers for LUAD.

Fig. 1

CHAF1B mRNA is up-regulated in LUAD. A The GEPIA2 database shows CHAF1B mRNA expression in a variety of malignant tumor types. B, C GEPIA2 and UALCAN databases showed that CHAF1B mRNA was up-regulated in LUAD. D The UALCAN database revealed a correlation between CHAF1B mRNA expression and TP53 mutation. E ROC curve of differentially expressed CHAF1B in patients with LUAD. F The GEPIA2 database showed the survival curve of differentially expressed CHAF1B in patients with LUAD. * P < 0.05

3.2 CHAF1B expression correlates with clinicopathological parameters in patients with LUAD

We examined the relationship between CHAF1B expression and clinicopathological characteristics of patients with LUAD to investigate the clinical implications of high CHAF1B expression in LUAD. The findings demonstrated that the overexpression of CHAF1B was positively correlated with the clinicopathological stage (I, II and III, IV), N stage (N0, N1 and N2, N3), and T stage (T1 and T2) of LUAD, with higher expression of CHAF1B in patients at a later stage (Fig. 2A–C). Additionally, we examined the prognostic correlation between CHAF1B and patients with LUAD. The findings indicated that in patients with stage I and II, N0, and M0 stages, the shorter the survival time, the greater the expression of CHAF1B (P < 0.05) (Fig. 2D–F). CHAF1B is crucial for LUAD onset and progression and has good prognostic value for patients with stage N0, M0, stage I and stage II disease.

Fig. 2

Expression of CHAF1B is related to the clinicopathological features of LUAD patients. A−C The differential expression of CHAF1B in different clinicopathological stages. A pathological grade. B N stage. C T stage. D–F the correlation between CHAF1B and the prognosis of LUAD patients with different clinical stages. D Stage I and II. E N0. F M0. *P < 0.05,**P < 0.01

3.3 The co-expression pattern of CHAF1B in LUAD

We searched for genes co-expressed with LUAD and produced a volcano map to identify genes that can interact with CHAF1B in LUAD and facilitate the understanding of the mechanism of CHAF1B (Fig. 3A). The top 50 most important genes that were positively or negatively correlated with CHAF1B (Fig. 3B, C) are also shown.

Fig. 3

Results of CHAF1B co-expression analysis. A Volcano map, red points indicate positive correlation of genes, and blue points indicate negative correlation of genes. B The heat map showed the first 50 genes positively correlated with CHAF1B. C Heatmap showed the top 50 genes negatively correlated with CHAF1B

3.4 The role of CHAF1B co-expressed genes in LUAD

We used pathway enrichment analysis to gain further insight into the mechanism of CHAF1B co-expressed genes in LUAD. According to the Metascape website, the top 100 genes co-expressed with CHAF1B were primarily clustered in the mitotic cell cycle, cell cycle regulation, chromosome organization, and cell cycle checkpoints (Fig. 4A, B). According to GO enrichment analysis, CHAF1B was mostly engaged in cellular processes, regulation of biological processes, and metabolic processes. (Fig. 4C). The PPI network of the top 100 genes co-expressed with CHAF1B was mainly concentrated in the mitotic cell cycle, mitotic cell cycle process, and the cell cycle (Fig. 4D, E). Additionally, MCODE analysis indicated that CHAF1B and its adjacent genes may affect ATR activation in response to replication stress, activation of the pre-replication complex, and DNA replication (Fig. 4F, G). In conclusion, these findings suggest that genes co-expressed with CHAF1B are mostly involved in the mitotic cell cycle and cell cycle activities in LUAD. Based on these findings, we hypothesized that CHAF1B is involved in the regulation of cell proliferation.

Fig. 4

CHAF1B and co-expressed genes in LUAD were analyzed using GO and PPI. A & B Cluster pathway analysis of genes co-expressed with CHAF1B. C CHAF1B involved in GO analysis bar chart. D & E CHAF1B and its adjacent genes involved in the protein–protein interaction network. F & G CHAF1B and adjacent genes involved in MCODE components

3.5 Analysis of CHAF1B at the single cell level in LUAD

The HPA database showed that the top three single cells with CHAF1B expression in lung cancer tissues were alveolar cells type 2, macrophages, and club cells. CHAF1B is mainly expressed in alveolar cells type 2, with 14.0 standardized transcripts per million protein-coding genes (nTPM) in this cell (Fig. 5A). Simultaneously, owing to the different expression levels of CHAF1B, each cell was divided into different types of cell clusters. CHAF1B is primarily expressed in c-8 cell clusters (Alveolar cells type 2) (Fig. 5B). We analyzed the correlation between the expression level of CHAF1B and the biological function of single LUAD cells using the CancerSEA database. The results showed that CHAF1B expression was positively correlated with DNA repair and the cell cycle, and negatively correlated with quiescence, inflammation and differentiation (Fig. 5C). These results further reveal the potential biological function of CHAF1B in LUAD at the single-cell level.

Fig. 5

Single-cell level analysis of CHAF1B in LUAD. A HPA database showed the single cell expression level of CHAF1B in lung cancer tissues. B The expression level of CHAF1B in different cell clusters of lung cancer. C CancerSEA database shows the correlation between CHAF1B and LUAD single cell biological function. *P < 0.05,**P < 0.01,***P < 0.001

3.6 The correlation between CHAF1B and immune infiltration in LUAD was analyzed

This study investigated the association between CHAF1B expression and immune cell infiltration in patients with LUAD. Spearman correlation analysis was conducted to examine the relationship between CHAF1B expression and immune cell infiltration, as well as immune checkpoints. The results indicated a significant positive correlation between CHAF1B expression and Th2 cell infiltration (R = 0.543, P < 0.001), and negative correlations with CD8 + T cell infiltration (R = −0.232, P < 0.001), B cell infiltration (R = − 0.241, P < 0.001), iDC cell infiltration (R = − 0.258, P < 0.001), and DC cell infiltration (R = − 0.218, P < 0.001) (Fig. 6A–F). Furthermore, there was a significant positive correlation observed between CHAF1B expression and the immune checkpoints PDCD1 (R = 0.127, P = 0.003), CD274 (R = 0.189, P < 0.001), LAG3 (R = 0.223, P < 0.001), and SIGLEC15 (R = 0.145, P < 0.001) as depicted in Fig. 7A–E. These findings suggest that CHAF1B may influence the tumor immune microenvironment and potentially serve as a novel target for immunotherapy in patients with LUAD.

Fig. 6

LUAD immune cell infiltration and CHAF1B relationship. A A comparison of 24 different immune cell types' infiltration and CHAF1B expression. B−F The correlation scatter plot between immune cell infiltration and CHAF1B expression. B Th2 cells. C CD8 T cells. D B cells. E iDC cells. F DC cells

Fig. 7

Immune checkpoints and CHAF1B correlate in LUAD. A The heat map demonstrating association between seven different types of immunological checkpoints and CHAF1B expression. B−E CHAF1B expression and immune checkpoint correlation scatter plot. B PDCD1. C CD27. D LAG3. E SIGLEC15

3.7 Correlation analysis between CHAF1B and drug sensitivity

In the CellMiner database, CHAF1B expression was significantly associated with the sensitivity of 12 FDA-approved drugs (Fig. 8). The CHAF1B expression level was positively correlated with the half maximal inhibitory concentration (IC50) of Nelarabine (Fig. 8A), Vorinostat (Fig. 8B), Methylprednisolone (Fig. 8C) and 6-Thioguanine (Fig. 8i). This finding suggests that patients with high CHAF1B expression may enhance Nelarabine, Vorinostat, Methylprednisolone and 6-Thioguanin resistance. In contrast, the expression level of CHAF1B was negatively correlated with IC50 of Alectinib (Fig. 8D), Defactinib (Fig. 8E), Celecoxib (Fig. 8F), Elesclomol (Fig. 8G), PF-06463922(lorlatinib) (Fig. 8H), Isotretinoin (Fig. 8J), brigatinib (Fig. 8K) and Olaparib (Fig. 8L).

Fig. 8

Correlation analysis between CHAF1B expression and drug sensitivity. A–L Correlation between CHAF1B expression and drug sensitivity scatter plot. A Nelarabine. B Vorinostat. C Methylprednisolone. D Alectinib. E Defactinib. F Celecoxib. G Elesclomol. H PF-06463922(lorlatinib). I 6-Thioguanine. J Isotretinoin. K brigatinib. L Olaparib

3.8 Construction of LncRNAs-miRNA-CHAF1B network in LUAD

Some lncRNAs can act as competitive endogenous RNA (ceRNAs) to competitively adsorb onto common sites of miRNAs and mRNA, thereby regulating mRNA expression [24, 25]. To further explore the molecular mechanism underlying the biological function of CHAF1B, we predicted the lncRNAs-miRNA-CHAF1B network using software tools. The ENCORI, miRWalk,TargetScan, DIANA-miroT, and miRcode databases were used to identify target miRNA of CHAF1B. These databases identified 18, 1711,700, 77, and 22 miRNAs, respectively. A total of 495 miRNAs were identified at the intersections of at least two databases (Fig. 9A). We selected three miRNAs that may be negatively correlated with mRNA expression, namely hsa-miR-29c-3p, hsa-miR-145-5p, and hsa-miR-1247-5p (Fig. 9B–D). According to the results shown in the ENCORI database, we found that hsa-miR-29c-3p, hsa-miR-145-5p, and hsa-miR-1247-5p were down-regulated in cancer tissues of LUAD patients (Fig. 9E–G). Next, we used the ENCORI database to predict the target lncRNA of miRNA and made a Sankey diagram. The findings indicated that six lncRNAs (AL13928711, AC016717.2, MIR4458HG, AC007036.3, NEAT1, and OIP5-AS1) might be the target lncRNAs of hsa-miR-29c-3p, NEAT1 and SNHG22 might be the target lncRNAs of hsa-miR-1247-5p, and three lncRNAs (SNHG1, MALAT1, AC006064.5) might be the target lncRNAs of hsa-miR-145-5p (Fig. 9H). Among them, AL139287.1, NEAT1 and SHG1 were highly expressed in cancer tissues of LUAD patients. They are more likely to be the lncRNA molecules that regulate the expression of CHAF1B (Fig. 9I–K).

Fig. 9

Construction of LncRNAs-miRNA-CHAF1B network in LUAD. A ENCORI database, miRWalk database,Targetscan database, DIANA-miroT database and miRcode database were used to identify the Wayne diagram of miRNAs binding to CHAF1B. B−D CHAF1B and target miRNA correlation scatter plot. B hsa-miR-29c-3p. C hsa-miR-145-5p. D hsa-miR-1247-5p. E–G Expression level of miRNA in cancer tissues and normal tissues of LUAD patients. E hsa-miR-29c-3p. F hsa-miR-145-5p. G has-miR-1247-5p. H Predicted Sankey diagram of lncRNAs-miRNA-CHAF1B network. I–K Expression level of lncRNA in cancer tissues and normal tissues of LUAD patients. I AL139287.1. J NEAT1. K SHG1. *P < 0.05,**P < 0.01,***P < 0.001

3.9 CHAF1B promotes the proliferation, migration, invasion and glycolysis of LUAD cells

The bioinformatics analyses provided a preliminary understanding of CHAF1B’s biological functions in LUAD. To validate our results, we used 13 clinical samples of LUAD cancer tissues and adjacent tissues to detect CHAF1B mRNA expression in lung adenocarcinoma tissues using qRT-PCR. Our findings demonstrated that lung cancer tumors expressed more CHAF1B mRNA than normal tissues (Fig. 10A). Additionally, CHAF1B was highly expressed in A549, H1975, and 95D cells relative to 16HBE cells with statistical significance observed in in A549 and H1975 cells (Fig. 10B). Given the high expression of CHAF1B in both LUAD tissues and cells, and its association with poor prognosis as indicated by bioinformatic analysis, we hypothesized that CHAF1B plays a significant influence in cancer progression in LUAD. To verify this hypothesis, we used si-CHAF1B to knock down CHAF1B mRNA expression in A549 and H1975 cells. We also constructed three siRNA-transfected cell lines to ensure knockdown efficiency. In A549 and H1975 cells, si-CHAF1B1 and si-CHAF1B3 demonstrated higher knockdown efficiencies in both cell lines (Fig. 10C, D). Thus, we selected si-CHAF1B1 and si-CHAF1B3 to transfect A549 and H1975 cells respectively. Using the CCK8 test, we discovered that the cell viability of si-CHAF1B3 and si-CHAF1B1 groups was reduced in A549 and H1975 cells (Fig. 10E−F). Transwell migration and invasion assays indicated that the knockdown of CHAF1B3 and CHAF1B1 resulted in decreased migration and invasion of A549 and H1975 cells (Fig. 10G−H). Furthermore, CHAF1B knockdown led to decreased glucose consumption and lactate metabolism in A549 and H1975 cells (Fig. 10I–L). Therefore, the knockdown of CHAF1B may inhibit the proliferation, migration, invasion, and glycolysis of A549 and H1975 cells.

Fig. 10

CHAF1B promotes the proliferation, migration, invasion and glycolysis of LUAD cells. A CHAF1B mRNA expression in LUAD along with adjacent tissues was found with qRT-PCR. B The expression of CHAF1B mRNA in A549, H1975 and 95D cells was detected by qRT-PCR. C−D qRT-PCR was used to detect the interference effects of si-CHAF1B1, si-CHAF1B2 and si-CHAF1B3. E−F CCK8 was used to determine the effect of CHAF1B on the proliferation of LUAD cells. G–H Transwell assay was used to detect the effects of CHAF1B on the migration and invasion ability of LUAD cells. I–L Glycolysis assay was used to detect the effect of CHAF1B on the glycolysis of LUAD cells.*P < 0.05,**P < 0.01,***P < 0.001,****P < 0.0001