High IKBIP expression as a biomarker for poor prognosis and correlated with immune inltrates in hepatocellular carcinoma (HCC)

Background: I kappa B kinase interacting protein (IKBIP) is dysregulated and closely correlated with prognosis in tumor. However, the potential functions of IKBIP have not been utterly revealed in hepatocellular carcinoma (HCC). Thus, we explored the clinical values of IKBIP and its correlation with immune inltrates in HCC. Methods: IKBIP expression in different tumor types was analyzed through the Tumor Immunity Evaluation Resource (TIMER) database. IKBIP expression between HCC and normal tissues were readily available for retrospective analyses from GEPIA, UALCAN and the Cancer Genome Atlas (TCGA) databases. The association between clinical features and IKBIP expression in HCC was analyzed by the Wilcoxon signed-rank test and logistic regression. Human Protein Atlas (HPA) was used to analyze the protein expression of IKBIP. A receiver operating characteristic (ROC) curve was performed to explore diagnostic potential of IKBIP. Kaplan-Meier (K-M) method and Cox regression were conducted to evaluate the inuence of IKBIP on the prognosis of HCC patients. Protein-protein interaction (PPI) networks were established using STRING. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for gene functional enrichment analysis. The interrelation between IKBIP expression and immune inltrates in HCC was analyzed through TIMER and tumor-immune system interaction database (TISIDB). Results: Our results revealed that IKBIP expression was abnormally upregulated in HCC, and ROC curve analysis conrmed the diagnostic power of IKBIP. High IKBIP expression was related to tumor sizes, pathologic stage and histologic grade. K-M plotter analysis showed that high IKBIP expression was correlated with poor prognosis of HCC patients. Moreover, multivariate Cox analysis further veried that high IKBIP expression was an independent risk factor in HCC patients. Correlation analysis found that IKBIP expression was associated with inltrating immune cells. Conclusion: High IKBIP expression is associated with poor prognosis and immune inltrates, which may serve as a biomarker target for HCC.


Introduction
Hepatocellular carcinoma (HCC), approximately accounting for 75%-85% of all primary liver malignancies, is one of the leading causes of cancer-related mortality around the world (1)(2)(3). In recent years, new targeted and immune therapies could improve overall survival and improve quality of life in HCC patients. However, the prognosis still remains unsatisfactory, and the 5-year survival rate is only 7% (4)(5)(6). Thus, more potential molecular targets are urgently needed for improving the treatment of HCC patients.
I kappa B kinase interacting protein (IKBIP) was found to be located on human chromosome 12. Hofer-Warbinek et al. showed that IKBIP was a target gene of p53 and promoted the apoptosis (7). A recent paper reported that IKBIP was upregulated and closely correlated with aggressive phenotypes in glioma.
High IKBIP expression was correlated with poor prognosis of patients with glioma. Moreover, IKBIP was closely related to a series of EMT-related biological processes, which indicated that IKBIP might contribute to tumorigenesis and progression of glioma through EMT induction(8). However, the potential functions of IKBIP in HCC still remain unclear. We hypothesized that, similar to glioma, the level of IKBIP is closely related to survival in HCC. Thus, we further explored the diagnostic and prognostic values of IKBIP in HCC and its correlation with immune in ltration based on multiple databases in this study.

Materials And Methods
Analysis of Tumor Immune Estimation Resource (TIMER) database TIMER (https://cistrome.shinyapps.io/timer/) is a comprehensive portal for the systematic assessment of immune in ltrations in different tumor types (9). In this study, IKBIP expression in diverse cancer types was analyzed via the "Diff Exp" module. Additionally, the "Gene" module was used to investigate the relationship between IKBIP expression and tumor-in ltrating immune cells (TILs).

Analysis of the Cancer Genome Atlas (TCGA) database
The IKBIP expression data and relevant clinical information in HCC patients were obtained from TCGA website (http://cancergenome.nih.gov/) (10). Besides, we eliminated any samples with duplicate or insu cient information in this study.

Analysis of Human Protein Atlas (HPA)
The protein expression of normal and tumor tissues was assessed via HPA (13). Thus, we used HPA to analyze the protein expression of IKBIP in HCC and tumor tissues.

Analysis of Kaplan-Meier (K-M) plotter database
The K-M plotter (http://kmplot.com/analysis/) can assess the effect of various biomolecules in tumor on the survival rate (14). In this study, the K-M plotter was performed to assess the prognostic potential of IKBIP in HCC patients.

Analysis of Protein-Protein Interaction (PPI) and gene functional enrichment analysis
The PPI network of IKBIP was established by STRING online database (https://string-db.org/) (15).
Meanwhile, Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for analyzing the possible biological functions of IKBIP.

Analysis of Tumor-Immune System Interaction database (TISIDB)
TISIDB is an online web for analyzing tumor-immune system interaction(16). We utilized TISIDB to analyze IKBIP expression and TILs in various cancers. Meanwhile, the interrelations between IKBIP expression and TILs were also evaluated by TISIDB.

Statistical Analyses
Mann-Whitney U-test and Paired t-test were performed to compare IKBIP expression levels between HCC and normal tissues. The ROC curve was conducted to assess the diagnostic potential of IKBIP. Wilcoxon signed-rank test and logistic regression were performed to analyze the correlation between clinical features and IKBIP expression in HCC. K-M plotter was used for assessing the in uence of IKBIP on survival. Univariate and multivariate Cox analyses were performed to explore the potential prognostic factors in HCC. The nomogram and calibration curves were conducted to predict 1-, 3-and 5-year overall survival (OS) for HCC patients. P value < 0.05 was considered as statistically signi cant.  Figure 1). Further studies showed that IKBIP expression in HCC patients was also elevated in GEPIA and UALCAN databases (Figure 2A-B). Moreover, we found consistent results in the TCGA database ( Figure 2C), and paired data analyses indicated that IKBIP expression in HCC tissues (n =50) was signi cantly higher than those in normal tissues (n = 50) ( Figure  2D). Besides, we found that IKBIP protein expression was upregulated in HCC tissue by immunohistochemical staining from HPA ( Figure 2E). In TCGA database, the ROC analysis suggested that the level of IKBIP expression had a diagnostic potential in HCC patients ( Figure 2F).

Discussion
HCC has become a major health problem worldwide with the characteristics of invasion, metastasis and frequent recurrence (17,18). The treatment of HCC has made great progress, but the prognosis of HCC still remains unsatisfactory (19). Thus, it is necessary to explore new immunotherapy targets for HCC. IKBIP is located on chromosome 12, which was described as the target of p53 with pro-apoptotic activity (20). A recent paper reported that IKBIP was upregulated in glioma and correlated with an unsatisfactory prognosis (21). However, the clinical values of IKBIP have not been utterly revealed in HCC.
In this study, our results found that IKBIP was elevated in various cancers by pan-cancer analysis. Moreover, we also con rmed that IKBIP expression in HCC was signi cantly upregulated in multiple databases. The IKBIP expression was associated with tumor sizes, high pathologic stage and histologic grade. ROC curve analysis found that IKBIP serves as a promising diagnostic biomarker in HCC patients. Based on K-M plotter and univariate analysis, we found that high IKBIP expression was related to short OS, PFS and DFS. Univariate Cox analysis further con rmed that the level of IKBIP was risk factors for the prognosis in HCC. These results indicated that IKBIP might serve as a novel prognostic factor and a potential new target for the treatment of HCC patients.
Recent studies have demonstrated that IKBIP could promote the occurrence and development of tumor by Epithelial-to-mesenchymal transition (EMT) induction(8). EMT plays a crucial role not only in promoting tumor invasion and migration, but also in tumor recurrence and treatment resistance (22,23). Besides, IKBIP has also been shown to be related to immune and in ammatory responses (24). Based on these, we conclude that IKBIP may play an immunosuppressive role in tumor. However, the biological function of IKBIP has not been investigated in HCC. Thus, we further elucidated the function of IKBIP in HCC. We found that Hub genes of IKBIP in PPI network were apoptotic protease-activating factor 1 (APAF1), GTPase-activating protein 3 (ARFGAP3), clathrin light chain B (CLTB), NFU1, protein kinase C and casein kinase substrate in neurons 2 (PACSIN2), patched domain containing 3 (PTCHD3), RAB11 family interacting protein 1 (RAB11FIP1), RPUSD4, serine/arginine-rich splicing factor 7 (SRSF7) and TTLL1. GO and KEGG analysis indicated that KIBIP and its co-expressed genes were enriched in endocytosis, Golgiassociated vesicle membrane, coated membrane, membrane coat and vesicle coat. Among co-expressed genes, ARFGAP3, a GTPase-activating protein, which was correlated with the Golgi-apparatus and involve in the vesicular tra cking pathway, and it has been con rmed that ARFGAP3 could promote the proliferation and migration of prostate cancer cell (25). RAB11FIP1, also known as Rab-coupling protein (RCP), which was involved in tumor invasion and metastasis in breast cancer(26). SRSF7 was frequently upregulated in colon and lung cancer, which supported that SRSF7 might serve as a proto-oncogene (27). These ndings indicated IKBIP might be involved in the occurrence and development of tumors by synergizing other tumor-related genes.
In recent years, the role of the immune system in cancer has been received increasing attention with the wide application of tumor immunotherapy (28,29). Accumulating studies demonstrated that immune cells in the tumor microenvironment (TME) contribute to tumor occurrence and progression (30). Therefore, we explored the interrelation between IKBIP expression and TILs. In this study, we found that IKBIP expression was closely correlated with to six tumor in ltrating immune cells (B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, dendritic cell) in HCC. Besides, we also found IKBIP expression was positively correlated with Act-CD4 + T cells, Tem-CD4 + T cells, NKT cells and Th2 cells. Our results suggested that there was a potential interrelation between IKBIP and immune in ltration cells in HCC.
Nevertheless, relevant research should be explored to verify the above ndings in future studies.
There are some limitations to address in this study. First, IKBIP expression and its prognostic value were analyzed by online public databases, which should be required to be validated in large-scale clinical samples. Second, the vivo/vitro experiments are needed to further explore the in uence of IKBIP on the immune in ltrations in HCC.
In conclusion, we rst found that IKBIP expression was upregulated and correlated with tumor sizes, pathologic stage and histologic grade in HCC. Moreover, IKBIP expression was also closely associated with the poor prognosis and immune in ltrations. Therefore, our ndings suggested that IKBIP could serve as a potential biomarker for poor prognosis and might play a crucial role in immune in ltrations in HCC. IKBIP expression in various types of cancers in TIMER database. *P < 0.05; **P < 0.01; ***P < 0.001.