RHBDF2 is correlated with immune infiltrates in hepatocellular carcinoma and may have potential as a biomarker

Immune checkpoint inhibitors have been approved for the treatment of advanced hepatocellular carcinoma (HCC). However, immunotherapy requires the identification of suitable biomarkers to guide treatment. The gene for rhomboid 5 homolog 2 (IRHom2), which encodes the rhombus protease iRhom2, activates the MAP3K7‐dependent pathway and promotes hepatic steatosis. Thus, we hypothesized an involvement of this gene in HCC. We report that RHBDF2 expression is dramatically upregulated in HCC. RHBDF2 upregulation is associated with tumor stage, lymph node metastasis, tumor protein P53 mutation, and worse prognoses in HCC patients. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis enrichment analysis indicated that RHBDF2 was involved in immune signal pathways. Moreover, RHBDF2 expression was positively related not only to immune infiltration, but also to immune cell markers. Examination of several types of HCC infiltrated by immune cells revealed that the group with high expression of RHBDF2 showed the worst outcomes. Therefore, RHBDF2 may have potential as a novel biomarker for predicting prognosis and is related to immune infiltrates in HCC.

Immune checkpoint inhibitors have been approved for the treatment of advanced hepatocellular carcinoma (HCC). However, immunotherapy requires the identification of suitable biomarkers to guide treatment. The gene for rhomboid 5 homolog 2 (IRHom2), which encodes the rhombus protease iRhom2, activates the MAP3K7-dependent pathway and promotes hepatic steatosis. Thus, we hypothesized an involvement of this gene in HCC. We report that RHBDF2 expression is dramatically upregulated in HCC. RHBDF2 upregulation is associated with tumor stage, lymph node metastasis, tumor protein P53 mutation, and worse prognoses in HCC patients. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis enrichment analysis indicated that RHBDF2 was involved in immune signal pathways. Moreover, RHBDF2 expression was positively related not only to immune infiltration, but also to immune cell markers. Examination of several types of HCC infiltrated by immune cells revealed that the group with high expression of RHBDF2 showed the worst outcomes. Therefore, RHBDF2 may have potential as a novel biomarker for predicting prognosis and is related to immune infiltrates in HCC.
Regarding the mechanism of inflammation in tumors, immunotherapy as a new therapeutic approach has received increasing attention. Recently, immune checkpoint inhibitors were approved, including Nivolumab, which is an option for advanced HCC patients. However, immune checkpoint inhibitors show a lower objective response rate and survival rate [10][11][12]. Thus, there is an urgent need to explore new prognostic biomarker in HCC.
The gene for rhomboid 5 homolog 2 (RHBDF2) is a protein-coding gene that encodes rhombus protease, iRhom2, which participates in important biological processes [13][14][15]. RHBDF2 can be expressed in immune cells, heart and epithelial cells, and has an important impact on the disease by activating the ADAM metallopeptidase domain 17 (ADAM17) signaling pathway [16,17]. Several recent studies have suggested that RHBDF2 is significantly upregulated in tumors and has pro-tumor effects [18][19][20]. Molecular mechanism studies have shown that RHBDF2 activates the mitogenactivated protein kinase 7 (MAP3K7)-dependent pathway and promotes hepatic steatosis [21]. Furthermore, RHBDF2 activates tumor necrosis factor (TNF)-a, and epidermal growth factor receptor (EGFR) signaling and enhances lupus nephritis [22]. High RHBDF2 expression can induce enhanced EGFR, WNT and transforming growth factor (TGF)-b signaling, and was dramatically linked to poor prognosis of cervical cancer [23]. Additionally, RHBDF2 overexpression activated TGF-b signaling and enhanced the aggressiveness of cancer cells in gastric cancer [24]. Recent studies have shown that RHBDF2 has a critical impact on the immune regulation of disease [16,25]. Therefore, RHBDF2 has a multifaceted influence on the tumor immune microenvironment, which has a pro-tumor effect. Nevertheless, the potential value and molecular mechanism of RHBDF2 in HCC patients are still unknown.
The present study aimed to evaluate the potential prognoses of RHBDF2 and its biological functions in HCC. Accoridingly, we evaluated RHBDF2 expression and its association with prognoses using bioinformatics tools. In addition, mutations of RHBDF2 and the link between RHBDF2 and immune cell infiltration were investigated in HCC. Mechanistically, we mined RHBDF2 co-expressed genes and analyzed their biological functions using bioinformatics tools. Importantly, quantitative reverse transcriptase-PCR, western blotting and immunohistochemistry were applied to detect RHBDF2 expression in HCC. Our data implied that the high RHBDF2 expression can predict a worse prognosis and regulate immune cell infiltration in HCC.

Analysis of RHBDF2 expression
We investigated RHBDF2 mRNA levels among 23  Cell culture, RNA extraction and quantitative reverse transcriptase-PCR (RT-qPCR) The normal human liver cell line LO2 and human HCC cell lines (Hep G2, SK-HEP-1, Hep 3B2.1-7) were provided by Professor Li Tang (Professor of Chongqing Medical University, Chongqing, China) and maintained in Dulbecco's modified Eagle's medium or RPMI1640 medium (Hyclone, Logan, UT, USA) with fetal bovine serum and penicillin and streptomycin (Beyotime Biotechnology, Shaghai, China). The incubator was set at 37°C with 5% CO 2 . The Trizol method (Takara, Kusatsu, Japan) was applied for extracting total RNA. The PrimeScript Reverse Transcription Kit (Takara) was employed for the synthesis of cDNA, and then RT-qPCR was carried out, and beta-actin expression was used to normalize the results. Beta-actin (forward primer: GCCCTAGACTTCGAGCAAGA; reverse primer: AGGAAGGAAGGCTGAA GAG), RHBDF2 (forward primer: GATGGGGCAGACACGTTTGA; reverse primer: CCTCGGAAGTAGCTGGCAG) were used for RT-qPCR.

Western blotting
After extracting the protein from the cells, we measured the protein concentrations, and then electrophoresed proteins. Subsequently, we transferred proteins to poly(vinylidene difluoride) membranes (Millipore, Burlington, MA, USA). We blocked membranes with non-fat milk and incubated membranes with RHBDF2 (dilution 1 : 1500; Proteintech Wuhan Sanying, Wuhan, China) and beta-actin (dilution 1 : 8000; Proteintech Wuhan Sanying) antibody. After washing, a secondary antibody was applied for incubating the membranes. Finally, the blots were detected with BeyoECL plus (Millipore), and the signals were visualized via a Gel Imaging System (Bio-Rad, Hercules, CA, USA).
in accordance with the Declaration of Helsinki. The study passed the ethical review of the Ethics Committee of Chongqing Medical University (permit number: 2022-K511). Briefly, after dewaxing, all tissue slices were rehydrated, antigen retrieved, and then 3% hydrogen peroxide was applied. Sections were incubated with RHBDF2 (dilution 1 : 200; Proteintech Wuhan Sanying) antibody at 4°C overnight after the block. A secondary antibody was applied to incubate the sections. Finally, 3,3 0 -diaminobenzidine was employed for staining and then sections were counterstained with hematoxylin. Two independent pathologists analyzed immunohistochemistry staining. When a disagreement occurred, a third senior pathologist was attended to make the final diagnosis [26]. After dewaxing, tissue slices were rehydrated and stained with hematoxylin and eosin, and then dehydrated and mounted.

Prognostic analysis
Kaplan-Meier Plotter (https://kmplot.com/analysis/index. php?p=background), a prognostic analysis database, was applied for investigating the prognosis of HCC patients in high-and low-expression RHBDF2 groups.
The clinical data for patients with HCC were downloaded from the Sanger box platform (http://vip.sangerbox. com) and then the link between RHBDF2 and the prognosis was evaluated. In addition to this, the prognostic effect of RHBDF2 was analyzed for different clinicopathological parameters of HCC. Moreover, the prognosis of patients with RHBDF2 overexpression based on immune cell infiltration was also explored.

Mutation analysis
The cBioPortal database (http://www.cbioportal.org), an online cancer genomics dataset, was employed for investigating the mutations of RHBDF2 in 1045 HCC patients from three datasets through the cBioPortal database. Apart from this, the prognostic values of HCC patients with RHBDF2 mutations or without RHBDF2 were no different.

Immune infiltration
The CIBERSORT database (https://cibersort.stanford.edu) contains immune data about various immune cell infiltration in various human cancers and can provide an estimate of the abundance of 22 tumor-infiltrating immune cells. We used this tool to examine the fraction of 22 immune cell types in HCC and normal tissues.
To investigate immune infiltration of HCC, we utilized the two latest algorithms, TIMER and Quan Tiseq [27], to measure the immune infiltration levels in 371 HCC patients. Furthermore, we evaluated the link between RHBDF2 upregulation and immune checkpoint molecules and markers of different immune cell subgroups with the TIMER database.

Statistical analysis
The GEPIA, UALCAN and TIMER databases were utilized for evaluating RHBDF2 expression, and the comparisons of RHBDF2 expression between HCC and normal tissues were performed with Student's t-test. Logistic regression was employed for evaluating the prognosis, and Pearson correlation analysis was also employed for evaluating the relationship between RHBDF2 expression and other gene expressions. P-value < 0.05 was considered statistically significant.

RHBDF2 expression is upregulated in HCC patients
First, the mRNA level of RHBDF2 was investigated in human cancers with the TIMER database, and we found that RHBDF2 mRNA expression was upregulated in most human tumors, including HCC (LIHC; Fig. 1A). The UALCAN database was utilized for further investigation of RHBDF2 expression in each tumor and corresponding normal tissues (Fig. 1B), which was consistent with the analysis of the TIMER database. Here, RHBDF2 expression in HCC was the main focus of the research. We observed that RHBDF2 mRNA expression was higher in HCC tissues than in normal tissues with the GEPIA and UALCAN databases (Fig. 1D). Furthermore, the CCLE database was employed for analyzing RHBDF2 expression levels of various HCC cell lines (Fig. 1C).

Association between RHBDF2 expression and clinical parameters in HCC
The UALCAN database was applied for investigating the link between RHBDF2 and the clinicopathological parameters in HCC. According to the tumor stage, RHBDF2 overexpression in HCC patients in stages 1, 2, and 3 was observed compared to normal liver tissue (Fig. 1E). Based on tumor grade, significant overexpression of RHBDF2 was observed in HCC patients in grades 1, 2, and 3 compared to normal liver tissue. Importantly, a clear difference in RHBDF2 upregulation was found in HCC patients in grades 1 and 3 ( Fig. 1F). For lymph node metastasis, significant overexpression of RHBDF2 was found in HCC patients with NO state compared to normal controls (Fig. 1G). Additionally, we found that RHBDF2 expression was upregulated in both TP53 mutant and TP53 wild-type HCC patients compared to normal controls, whereas RHBDF2 expression was significantly higher in TP53 mutant HCC patients than in TP53 wild-type HCC patients (Fig. 1H). The results above indicate that high RHBDF2 expression remarkably correlated with progression and lymph node metastasis in HCC patients.

Validation of differential expression of RHBDF2 in HCC
To analyze in-depth protein expression of RHBDF2 in HCC, protein expression of RHBDF2 in HCC and normal tissues was examined by immunochemistry staining. RHBDF2 overexpression was discovered in HCC according to the results of immunochemistry and was mainly expressed in the cytoplasmic compared to normal tissues (Fig. 2C,D). It is worth noting that RHBDF2 expression was linked to the differentiation degree of HCC tissues (r = 0.438, P = 0.005) ( Table 1). The results of hematoxylin and eosin staining in HCC and adjacent tissues are shown in Fig. 2A,B. Additionally, the expression of RHBDF2 also was examined in several HCC cell lines. We discovered that RHBDF2 expression is upregulated in HCC cell lines (HepG2, SK-Hep1, Hep3B2.1-7) according to the outcomes of RT-qPCR and western blotting compared to normal liver cell line LO2 (Fig. 2E,F).

Correlation between RHBDF2 expression and prognosis of HCC patients
For an understanding of how RHBDF2 exerted influence on the prognosis of HCC patients, we first applied the Kaplan-Meier database for evaluating the link between RHBDF2 and prognosis in HCC patients. We observed that RHBDF2 overexpression significantly correlated with poorer overall survival (OS) [hazard ratio (HR) = 1.57, P = 0.011], progression-free survival PFS (HR = 1.6, P = 0.0071) and recurrence-free survival (RFS) (HR = 1.55, P = 0.0041) in HCC patients, but not with disease-specific survivial (DSS) (HR = 1.48, P = 0.096) (Fig. 3A-D). To evaluate the prognostic effect of RHBDF2, the clinical data of HCC were downloaded and a prognostic analysis of RHBDF2 overexpression was performed. We found that the outcomes are consistent with the analysis of the Kaplan-Meier plotter (Fig. 3E-H). Therefore, these results implied that there is an obvious relationship between RHBDF2 upregulation and worse prognosis in HCC. The Kaplan-Meier plotter was employed for investigating the link between RHBDF2 expression and different clinicopathological characteristics in HCC to further explore the prognostic function of RHBDF2 in HCC patients. For the tumor stage, RHBDF2 overexpression was associated with unfavorable DSS in stage 1 HCC patients and related to bad OS and RFS in stage 3 HCC patients (Fig. 4A,C,D). In addition, RHBDF2 upregulation was related to bad OS in American Joint Committee on Cancer (AJCC) stage T-1 HCC patients, and related to unfavorable OS, bad RFS, poor PFS, poor DSS in AJCC stage T-2 HCC patients, and associated with poor RFS in AJCC stage T-3 HCC patients (Fig. 4A-D). For the vascular invasion, the results showed that RHBDF2 upregulation was related to unfavorable RFS and PFS in HCC patients with no vascular invasion (Fig. 4B,C). For the tumor grade, RHBDF2 overexpression was linked ed to unfavorable OS in stage 1 HCC patients and poor RFS in grade 3 HCC patients, and was related to worse OS, PFS, RFS and DSS in stage 2 HCC patients (Fig. 4A-D). For the metastasis stage, RHBDF2 overexpression was related to unfavorable OS, PFS and DSS in HCC patients with M0, but related to good PFS and DSS in HCC patients with MX (Fig. 4A,C,D). For the lymph node metastasis stage, RHBDF2 upregulation was linked to the bad OS and DSS in HCC patients with the N0 stage but related to good DSS in HCC patients with NX stage (Fig. 4A,C,D). Notably, the status of MSI does not influence the prognosis of HCC patients (Fig. 4). Moreover, a clear association between RHBDF2 upregulation and worse OS and poor PFS  was found in male HCC patients and poor RFS was observed in female HCC patients (Fig. 4A-C). Interestingly, a significant relationship between RHBDF2 overexpression and worse OS, poor RFS, unfavorable PFS and worse DSS was found in white and Asian patients with HCC (Fig. 4A-D). Additionally, RHBDF2 overexpression was clearly related to unfavorable OS, PFS, RFS and DSS in drinking HCC patients (Fig. 4A-D).
Notably, we found that there is a noticeable link between RHBDF2 and worse RFS and PFS in HCC patients with a hepatitis history, and poor OS and DSS in HCC patients without a hepatitis history (Fig. 4A-D). Therefore, these results demonstrated that RHBDF2 has prognostic value in HCC patients.

Mutation analysis of RHBDF2 in HCC
To analyze the alteration frequency of RHBDF2 in HCC, 1045 patients in total with HCC were investigated using the cBioPortal database (LIHC, AMC, Hepatology 2014; LIHC, TCGA, Firehose Legacy; LIHC, TCGA, PanCancer Atlas). The alteration frequency of RHBDF2 was 5.84%, 3.49% and 0.43% in these three datasets, respectively (Fig. 5A,B). The most obvious type was amplification (Fig. 5A,B). We found four mutation sites (including three missense and one inframe mutation) between amino acids 128 and 334 (Fig. 5C). However, a statistical difference in OS, PFS, disease-free survival (DFS) and DSS in HCC patients with or without RHBDF2 alterations was not observed with the Kaplan-Meier plotter (Fig. 5D).

GO, KEGG and GSEA pathway analysis of RHBDF2 in HCC
Importantly, RHBDF2 co-expressed genes were mined to evaluate the biological function of RHBDF2 in HCC using the LinkedOmics database (Fig. 6A). We showed the top 50 genes significantly correlated with RHBDF2 (Fig. 6B,C). Then, the top 500 genes positively linked to RHBDF2 were utilized for enrichment analysis of GO and KEGG to explore the biological value of RHBDF2. Subsequently, the top 10 enriched terms in GO and KEGG enrichment were obtained (Fig. 6D,E). Notably, GO biological process enrichment analysis indicated that RHBDF2 was related to some immune signaling pathways, including positive regulation of immune response, interleukin-2 production and the cytokine-mediated signaling pathway (Fig. 6D). Regarding KEGG pathway enrichment analysis, RHBDF2 was also associated with immune pathways, such as the TNF signaling pathway and cytokine-cytokine receptor interaction (Fig. 6E).
To obtain more insight into the molecular mechanism of RHBDF2 in HCC, GSEA analysis was carried out on the RHBDF2 upregulation group and the RHBDF2 downregulation group based on gene expression data sets. The enriched terms in KEGG (Fig. 6F,G) were also revealing. We also discovered that RHBDF2 upregulation was related to multiple immune pathways in HCC according to KEGG pathway enrichment analysis. Therefore, the above results implied that RHBDF2 has important consequences in immune pathways in HCC.

Association between RHBDF2 expression and immune infiltrates
Lymphocyte infiltration was an important independent predictor for relapse in HCC patients [28]. Therefore, 22 immune cell types in HCC and normal tissues were first analyzed with the CIBERSORT database. We discovered that immune cell infiltration was stronger in HCC compared to normal tissues (Fig. 7A). The Quan TIseq tool was utilized to investigate the association between RHBDF2 upregulation and infiltration of varied immune cells in HCC. A significant relationship between RHBDF2 upregulation and high immune cell infiltration was observed in HCC (Fig. 7B). Apart from this, the TIMER database was applied for evaluating the link between RHBDF2 upregulation and the infiltration of immune cells in HCC. The findings showed that the RHBDF2 upregulation was positively correlated with the infiltration of different immune cells in HCC. This was consistent with the analysis of the Quan TIseq tool (Fig. 7C). Cancer immune escape influences oncological outcomes and survival of cancer patients. Immune checkpoint molecules, such as PD-1 and PD-L1, can promote tumor cells to escape immune surveillance. To evaluate the association between RHBDF2 and the tumor microenvironment in HCC tissues, we investigated the link between RHBDF2 and immune checkpoint genes. Notably, a strong correlation between RHBDF2 overexpression and immune checkpoint gene upregulation was observed with the TIMER and GEPIA databases (Fig. 7D). These findings imply that RHBDF2 has important consequences for immune escape in the HCC microenvironment.

Association between RHBDF2 expression and various immune markers
For a deeper understanding of the relationship between RHBDF2 overexpression and immune infiltration in HCC patients, we evaluate the correlation between RHBDF2 overexpression and gene markers of immune cells with the TIMER database. The outcomes showed that RHBDF2 was clearly linked to most gene markers in HCC patients (Table 2). Additionally, the relationship between RHBDF2 upregulation and gene markers of various functional T cells was assessed with the TIMER database. An obvious relationship between RHBDF2 overexpression and gene markers of various functional T cells was shown with the TIMER database (Table 3). It is noteworthy that the association between RHBDF2 expression and major histocompatibility complex (MHC) molecules was also investigated. We discovered that RHBDF2 upregulation was clearly related to the MHC molecular markers, and this correlation had statistical significance (Table 4). These results implied that RHBDF2 has an essential impact upon immune cell infiltration of HCC.

Prognostic analysis of RHBDF2 expression based on immune cells in HCC patients
The present study indicated that RHBDF2 upregulation was surely related to immune cell infiltration in HCC. In addition, RHBDF2 overexpression had an unfavorable prognosis for HCC patients. Thence, we conjecture that RHBDF2 can impact the prognosis in HCC patients partly because of immune cell infiltration. The Kaplan-Meier plotter was applied for performing the prognosis evaluation of RHBDF2 based on immune cell infiltration subgroups in HCC. The results showed that patients with high RHBDF2 expression and enriched natural killer T cells (P = 0.0031), Th1 cells (P = 0.023), mesenchymal stem cells (P = 0.0023), macrophages (P = 0.002) and regulatory T cells (P = 0.003) had a poor OS (Fig. 8C,D,F-H). However, there is no link between RHBDF2 overexpression and OS in HCC patients with enriched CD4 + memory T cell, CD8 + T cell and Th2 cell infiltration (Fig. 8A,B,E). Similarly, high RHBDF2 expression in HCC patients was associated with a worse RFS in enriched natural killer T cells (P = 0.014), Th1 cells (P = 0.0067), Th2 cells (P = 0.035), mesenchymal stem cells (P = 7.6E-05), macrophages (P = 0.00029) and regulatory T cells (P = 0.0037) ( Fig. 8K-P). However, we found no relationship between RHBDF2 expression and RFS in HCC patients with enriched CD4 + memory T cells and CD8 + T cells (Fig. 8I,J). The above results implied that RHBDF2 may have important consequences in the prognosis of HCC patients partly because of immune infiltration.

Discussion
HCC is a primary malignancy and its mortality and morbidity is increasing year-to-year [4,[29][30][31]. Surgical  [4,7,8,32,33]. Thus, it is important to explore a new prognostic biomarker in HCC urgently. RHBDF2 is a protein-coding gene that participates in important biological processes, consisting of regulation of ADAM17, TNF signaling, and EGFR activation [34,35]. For a detailed understanding, the biological roles and potential mechanism of RHBDF2 in HCC, as well as the expression, prognosis and mutation of RHBDF2, and its link with immune infiltrates in HCC, were first analyzed.
In the present study, significant RHBDF2 overexpression in HCC tissues was discovered using the TIMER, GEPIA, and UALCAN databases compared to normal tissues. These results indicated RHBDF2 was correlated with progression and had a pro-cancer influence in HCC. Subsequently, we further investigated RHBDF2 expression in HCC cell lines and HCC tissues. Immunohistochemical staining was utilized to investigate RHBDF2 expression in HCC tissues. Additionally, RT-qPCR and western blotting were employed for the evaluation of RHBDF2 expression in HCC cell lines. These outcomes indicated that RHBDF2 was remarkably upregulated in HCC cell lines and HCC tissues.
Additionally, we also assessed the link between RHBDF2 and clinicopathological parameters and found that RHBDF2 overexpression was significantly linked to tumor histology, stage, lymph node metastasis and TP53 mutation. These results are consistent with previous research. High RHBDF2 expression was positively associated with tumor stage, tumor size and extent of tumor invasion [23]. In addition, RHBDF2 alteration frequency in HCC was investigated with the cBioPortal database. The results suggested that HCC patients had alterations of RHBDF2, and the most obvious type was amplification. From the results of survival analysis, RHBDF2 upregulation was clearly related to worse OS, RFS and PFS, which is consistent with previous findings suggesting that RHBDF2 has a crucial influence on poor prognosis [23]. Our results implied that RHBDF2 was linked to the prognosis in HCC patients. Furthermore, we further researched the link between RHBDF2 and prognostic value in different clinicopathological parameters of HCC patients. Multivariate COX regression analysis suggested that RHBDF2 is a risk factor, which was consistent with survival analysis. Thus, these results implied that RHBDF2 can be a novel potential biomarker for the prediction of prognosis in HCC patients.
Immune escape and immune tolerance have important influences on tumor progression and immune infiltration is linked to tumor occurrence, development and metastasis as a result of HCC being a typical inflammation-related tumor [36][37][38]. Therefore, a comprehensive analysis of immune infiltrates in HCC patients was first carried out to reflect the changes in immune status in HCC patients. For CIBERSOT analysis, the infiltration of 22 immune cells was clearly increased compared to normal tissues. Previous studies have reported that RHBDF2 was involved in the immune process of disease. For example, the upregulation of RHBDF2 expression can promote the progression of cervical cancer by inducing TGF-b transcription [23], RHBDF2 promotes the transport and activation of ADAM17 is the key to activating tumor necrosis factor signaling [35], RHBDF2 can mediate inflammation by releasing TNF-a [22,[39][40][41][42] and downregulation of RHBDF2 expression can lead to changes of cytokine production in T helper cells [43]. Therefore, to evaluate the potential biological values of RHBDF2 in HCC, RHBDF2 co-expressed genes were applied for enrichment analysis of GO and KEGG. We found that RHBDF2 co-expressed genes were related to multiple pathways of the immune process with GO and KEGG enrichment analysis, which was consistent with the GSEA analysis. In many cancer patients, the lymphocyte infiltration rate can independently predict survival rate and lymphatic metastasis [44]. Lymphocyte infiltration rate predicts the recurrence in HCC patients [28]. Therefore, the link between RHBDF2 and immune infiltration was evaluated through correlation analysis, and RHBDF2 upregulation was likely related to multiple immune cell infiltration in HCC. Additionally, a strong correlation between RHBDF2 upregulation and immune checkpoint genes (PDCD1, CD274, LAG3, CTLA4, TIGIT, HAVCR2) was observed in HCC, and these immune checkpoint genes can contribute to tumor immune escape and promote tumor progression [45][46][47]. It is noteworthy that RHBDF2 upregulation was clearly associated with various immune cell markers, lymphocyte subset markers and MHC molecular markers in HCC. Therefore, the present study showed that RHBDF2 overexpression was indeed linked to the immune infiltrates in HCC patients, which indicated that RHBDF2 might have a crucial influence on the immune infiltration of HCC. More importantly, we observed that RHBDF2 overexpression in HCC with enriched immune cells had a worse prognosis through the analysis of the Kaplan-Meier plotter. Thus, RHBDF2 can impact the prognosis of HCC patients partly because of immune infiltration. The high density of Tregs is positively associated with vascular invasion and negatively related to tumor packaging [48] and the high density of Tregs is linked to the worse prognosis of HCC patients [49]. Macrophage promoted tumor growth and invasion and predicted poor prognosis in HCC [50][51][52][53][54][55][56]. These outcomes indicated that RHBDF2 can act as a new prognostic biomarker related to immune infiltration of HCC.
The present study can strengthen our comprehension of the function of RHBDF2 in HCC; that is, upregulated RHBDF2 is clearly linked to clinicopathological features, an unfavorable prognosis and immune infiltrate in HCC, although there are still some limitations. First, most of the investigation is based on the mRNA level of RHBDF2. An in-depth analysis based on the protein level is more convincing. Second, RHBDF2 may have an essential impact upon the immune regulation process of HCC, but its molecular mechanism needs more research. Overall, our findings implied that RHBDF2 overexpression can predict the worse prognosis of HCC patients. Furthermore, our research implied that RHBDF2 upregulation was significantly linked to immune cell infiltration and revealed a potential molecular mechanism by which that RHBDF2 can impact the prognosis of HCC patients partly because of tumor immune infiltration.

Conclusions
The present study systematically analyzed the expression, prognosis and immune infiltrates of RHBDF2 in HCC. It was implied that RHBDF2 overexpression was clearly linked to the unfavorable prognosis of HCC patients partly because of immune cell infiltration. Therefore, we concluded that RHBDF2 could be a new biomarker for the prediction of the prognosis of HCC patients.