Pan-cancer CENPF expression analysis
CENPF expression in the pan-cancer data from GTEx and TCGA was detected initially. The analysis exhibited the expression of CENPF was revealed to be elevated in 29 kinds of cancer, containing BRCA, BLCA, ACC, COAD, CHOL, CESC, GBM, ECSA, DLBC, KIRC, KICH, HNSC, LIHC, LGG, KIRP, OV, LUSC, LUAD, PRAD, PCPG, PAAD, SKCM, SARC, STAD, READ, THCA, TGCT, UCS and UCEC (Figure 1A-1D). In addition, CENPF was highly expressed in LUAD based on GSE75037, GSE32863 and GSE40791 (Figure 1E−1G). The analysis of UALCAN database showed that the expression of CENPF was up-regulated in the tumor tissues of LUAD patients as in contrast to the adjacent normal tissues (Figure 2A). Simultaneously, the CENPF expression level in LUAD increased with the progression of tumor grade (Figure 2B). Moreover, IHC outcomes in the HPA database exhibited that the expression of CENPF was evidently up-regulated in LUAD lungs in contrast to controls containing normal histology (Figure 2C). The CENPF expression was measured through qRT-PCR in H1299, A549, H1975 and PC9 cells. According to our findings, CENPF was overexpressed in H1299, A549, H1975 and PC9 cells in comparison with the HBE cells (Figure 2D-2F).
Association between CENPF expression and cancer patient prognosis
We explored the relationship between the expression of CENPF and PFS, DSS, OS and in different kinds of tumors utilizing GEPIA2 database and the TCGA cohort for determining CENPF value in the prediction of cancer patient prognosis. The outcomes indicated higher expression of CENPF was related to an evident decreased OS in LGG (p = 1.8e-05), KIRC (p = 0.0036), ACC (p = 0.00024), LUAD (with p value of 0.0098), LIHC (p = 0.0018), KIRP (with p value of 0.00046), MESO (p = 1.8e-07), SARC (p = 0.012), SKCM (p = 0.024; Figure 3A-3I). However, THKM (p = 0.0074) demonstrated a tendency toward better survival as CENPF expression increased (Figure 3J). Besides, Increased CENPF expression was correlated to a reduced DSS in KIRP (p = 3e-04), KIRC (p = 1e-04), ACC (p < 0.0001), LUAD (p < 0.0013), LIHC (p = 0.0063), LGG (p < 0.0001), SARC (p = 0.0403), PAAD (p = 0.0287), MESO (p < 0.0001), SKCM (p = 0.0023; Supplementary Figure 1). With higher CENPF expression, PFS decreased in the ACC (p = 2e-04), GBM (p = 7e-04), CESC (p = 0.0367), BLCA (p = 0.0405), LGG (p = 0.0012), KIRP (p < 0.0001), KIRC (p = 3e-04), MESO (p = 0.0021), LUAD (p = 0.025), LIHC (p = 9e-04), THCA (p = 0.0162), SARC (p = 0.0048), PRAD (p = 0.0024), UCEC (p = 0.0265) and UVM (p = 0.0036; Supplementary Figure 2).
Correlation and enrichment analysis
To ascertain the role of CENPF, we utilized TCGA data to conduct a correlation study between CENPF and other LUAD genes. For enrichment analysis, the first 300 genes remarkably associated with CENPF were chose (Supplementary Table 1). We next used Metascape to thoroughly investigate biological pathways with above 300 genes. Gene Ontology (GO) analysis revealed that mitotic cell cycle process, cell cycle process regulation together with microtubule skeleton organization were significantly enriched in BP; microtubule binding, ATP-dependent activity and chromatin binding were markedly abundant in MF; spindle, spindle pole, as well as chromosomal region were significantly enriched in CC (Figure 4A-4C). Moreover, the study of KEGG pathway suggested that DNA replication, fanconi anemia pathway and cell cycle were obviously abundant (Figure 4D). GSEA was employed to determine Reactome and KEGG pathways, which revealed that the progesterone-mediated oocyte maturation and the degradation of mitotic proteins pathways mediated by APC/C and APC/C:Cdc20 activation were significantly enriched (Figure 4E-4F). These findings showed that CENPF was participated in various pathways related to cancer in LUAD.
CENPF expression and immune cell infiltration analysis
Further investigation of LUAD immune cell infiltration score in TCGA revealed that the level of T cell CD4+ Th2 infiltration was substantially higher in the group with high CENPF expression (Figure 5A-5B). This result indicated that high expression CENPF promoted the infiltration level of T cell CD4+ Th2, which demonstrated a correlation with the immunosuppressive state in LUAD[22]. T cell CD4+ Th2 expression was significantly enhanced in tumor patients, which promoted the occurrence and development of malignancies by decreasing cellular immune function[23]. Likewise, we discovered that three indicators scored substantially lower on groups of high CENPF expression, including immune score, microenvironment score, and stroma score (Figure 5C-5E). As well, we further verified the significantly positive correlation between the T cell CD4 + Th2 infiltration level and CENPF expression in the LUAD tissues through Timer 2.0 database (Figure 6A).
Correlation between CENPF and immune checkpoint-related genes, TMB or MSI in LUAD
Subsequently, the significantly positive correlation between TMB/MSI score and the CENPF expression level in LUAD was found (Figure 6B-6C). Then, we analyzed the correlation between CENPF and immune checkpoint-related genes using LUAD data in TCGA and found that CENPF was also highly positively correlated with immune checkpoint-related genes in LUAD, such as CD274, KIR2DL3, LAG3, LARS1, NECTIN2, PDCD1, TGFBR1 and TIGIT (Figure 6D). Following these findings, it was hypothesized that increased CENPF expression was strongly associated with immunosuppressive state in patients with LUAD[24]. Moreover, several immune checkpoints were chosen with the aim of exploring differential expression levels between high CENPF expression and low CENPF expression, and found that CD274, LAG3, PDCD1 and TIGIT were substantially increased in the group with high CENPF expression (Figure 6E). These outcomes exhibited that high expression of CENPF was associated with tumor immunosuppression in LUAD patients. The results presented CENPF was positively correlated with many known immune checkpoints so as to inhibit the immune state of tumor. However, the score of TMB/MSI indicated that there may be potential immune checkpoints to be found and the immunosuppression status of LUAD could be effectively improved in patients with LUAD containing high CENPF expression.