Upregulated UBE4B expression correlates with poor prognosis and tumor immune infiltration in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is a major human health concern. Increasing evidence has demonstrated that ubiquitin ligase E4B (UBE4B) may be involved in the occurrence and development of various human cancers and may affect prognosis. However, the specific role and mechanism of UBE4B in HCC is unclear. Methods: A pan-cancer analysis of UBE4B expression, clinicopathological features, and prognosis was performed using bioinformatics techniques. Subsequently, the expression, prognosis, and correlation of UBE4B and its upstream miRNAs and lncRNAs were analyzed. We investigated the relationship between UBE4B expression and immune cell infiltration, immunomodulatory factors, and chemokines in HCC. The expression levels of UBE4B and its upstream lncRNAs (FGD5-AS1, LINC00858, and SNHG16) and miRNAs (hsa-miR-22-3p) were evaluated in HCC cell lines using qRT-PCR. Results: UBE4B expression increased in HCC and was correlated with a poor survival rate in patients with HCC. A ceRNA network was established to identify the UBE4B-hsa-miR-22-3p-FGD5-AS1/LINC00858/SNHG16 regulatory axis in HCC. UBE4B expression was significantly associated with immune cell infiltration, immunomodulators, chemokines, and their receptors in HCC. The mRNA expression of FGD5-AS1, LINC00858, SNHG16, and UBE4B was higher in the HCC cell lines (7721 and HepG2) than in the normal hepatocyte line (LO2), and the expression of hsa-miR-22-3p mRNA showed a decreasing trend. Conclusions: Our findings showed that upregulation of UBE4B was associated with poor prognosis and tumor immune infiltration in HCC. These findings will aid in understanding the relevant functions of UBE4B and provide new strategies for drug development and exploration of prognosis-related biomarkers.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common pathological type of liver cancer and the leading cause of death in patients with liver cancer [1,2]. The incidence and mortality rates of liver cancer worldwide rank 6 th and 3 rd of all cancers, respectively, with approximately 960,000 new cases and 830,000 deaths per year [3]. The main risk factors of HCC are hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcohol consumption, obesity, diabetes, non-alcoholic fatty liver disease, aflatoxins, etc. [4,5]. Although significant progress has been made in treating HCC with surgery, chemotherapy, targeted therapy, immunotherapy, and interventional therapy, the early symptoms of HCC are atypical, and most patients are at advanced stages when diagnosed. At this time, the available treatments are limited, resulting in a poor prognosis with a 5-year survival rate of approximately 18.4% [6,7]. Therefore, it is importance to explore more effective therapeutic targets and identify novel prognostic markers to improve HCC survival.
Protein post-translational modifications (PTMs) are processes that employ reversible state patterns to modulate protein function and alter physicochemical properties [8]. Studies have identified more than 400 post-translational modifications, with common modifications including ubiquitination, sumoylation, phosphorylation, acetylation, and methylation [9]. The ubiquitin-proteasome system (UPS) is a common posttranslational modification pathway that is involved in the regulation of cell survival and differentiation [10]. Ubiquitination requires the participation of three enzymes, namely ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2), and ubiquitin-protein ligase (E3) [11]. UBE4B belongs to the U-box family of ubiquitin ligases, also known as UFD2a, which is a novel E3 [12]. Many studies have investigated the substrate function of UBE4B, and it has been confirmed that it is involved in the ubiquitinated degradation of important proteins such as EGFR, p53, and caspase3 [13,14]. Additionally, UBE4B can be involved in the development of various cancers, such as renal cell carcinoma, breast cancer, and neural tube cell tumors, by regulating the ubiquitination of substrate proteins [15][16][17]. These results suggest that UBE4B may be a novel prognostic indicator in patients with multiple malignancies.
In this study, we investigated the UBE4B expression, prognosis, tumor immune microenvironment, and regulatory mechanisms of HCC. Next, we explored non-coding RNA regulation associated with UBE4B and constructed a critical UBE4B-miRNA-LncRNA regulatory axis. These findings will aid us to understand the relevant functions of UBE4B and provide new strategies for drug development and exploration of prognosis-related biomarkers.

Data acquisition
The expression of UBE4B was evaluated in 33 cancer and normal tissues using the The Cancer Genome Atlas (TCGA) (https://www.cancer.gov/tcga) and The Genotype-Tissue Expression (GTEx) (http://commonfund.nih.gov/GTEx/) databases. RNA sequencing data and clinical follow-up information of 33 patients with cancer were obtained from the TCGA cohort. All expression data were normalized using a log2 transformation.

TIMER database analysis
Tumor Immune Estimation Resource (TIMER) (https://cistrome.shinyapps.io/timer/) is a web server for analyzing the abundance of tumor infiltrates [18]. It was used to investigate the expression and abundance of immune infiltration in different cancers based on the TCGA cohort, with the threshold value as log2TPM.

GEPIA database analysis
Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) is a web tool for cancer and normal gene-expression profiling and interactive analysis using TCGA and GTEx cohorts [19]. Analysis of violin images of UBE4B in various pathological stages were performed using the Pathological Stage Map module in the GEPIA database. axis in HCC. UBE4B expression was significantly associated with immune cell infiltration, immunomodulators, chemokines, and their receptors in HCC. The mRNA expression of FGD5-AS1, LINC00858, SNHG16, and UBE4B was higher in the HCC cell lines (7721 and HepG2) than in the normal hepatocyte line (LO2), and the expression of hsa-miR-22-3p mRNA showed a decreasing trend. Conclusions: Our findings showed that upregulation of UBE4B was associated with poor prognosis and tumor immune infiltration in HCC. These findings will aid in understanding the relevant functions of UBE4B and provide new strategies for drug development and exploration of prognosis-related biomarkers. AGING

Kaplan-Meier plotter analysis
The relationship between UBE4B expression and prognosis, including overall survival (OS) and recurrence-free survival (RFS), was examined using the Kaplan-Meier plotter (http://kmplot.com/analysis/) in different cancer types. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated along with the log-rank p-value.

TISIDB database analysis
TISIDB (http://cis.hku.hk/TISIDB/index.php) is an online platform for the interaction between tumors and the immune system that integrates multiple heterogeneous data types [20]. The TISIDB database was used investigated the relationship between UBE4B and 45 immunostimulants, 24 immunosuppressive agents, 41 chemokines, and 18 receptors in HCC.

HPA database analysis
The Human Protein Atlas (HPA) (www.proteinatlas.org/) was used to detect the protein expression of UBE4B in hepatocellular carcinoma and corresponding normal tissues in the "tissue" and "pathology" modules. All images were confirmed by immunohistochemical experiments and specific patient information is listed.

Statistical analysis
All data analyses were automatically performed using the online database mentioned above. Log-rank p-value <0.05 or p-value <0.05 were considered statistically significant.

Availability of data and materials
All data generated or analyzed during this study are included in this article.

Forecast and evaluation of the upstream miRNAs of UBE4B
The miRNAs upstream of UBE4B was predicted using the TarBase V.8 database, and 55 miRNAs were retrieved ( Figure 3A). In accordance with the ceRNA hypothesis, we estimated the predicted miRNA expression patterns and prognostic value in LIHC using the starBase database. The results indicated that only downregulation of hsa-miR-22-3p was correlated with worse OS in patients with LIHC (P = 5.4 × 10 -10 ; P = 0.0013) ( Figure 3B, 3C). A correlation analysis of UBE4B and hsa-miR-22-3p was conducted ( Figure 3D), indicating that the hsa-miR-22-3p/UBE4B axis was involved in the ceRNA mechanism. Therefore, hsa-miR-22-3p could be considered as a key miRNA for follow-up exploration.

Verification of the mRNA and protein expression of genes
The mRNA expression levels of FGD5-AS1, LINC00858, SNHG16, hsa-miR-22-3p, and UBE4B were evaluated in HCC cell lines. The mRNA expression levels of FGD5-AS1, LINC00858, SNHG16,  and UBE4B were elevated in the HCC cell lines (7721 and HepG2) compared to the normal liver cell line (LO2), while the mRNA expression of hsa-miR-22-3p showed a decreasing trend ( Figure 7A-7E). Furthermore, we explored the protein expression of UBE4B in HCC using the HPA database. The results revealed that UBE4B protein expression was higher in HCC tissues than in corresponding normal tissues, mainly in the cytoplasm and cellular membranes ( Figure 7F-7I).

DISCUSSION
HCC has been a problem affecting human health for many years. There are observable variations in tumor proliferation, invasion, and metastasis between different HCC subtypes; therefore, it is necessary to further investigate biological molecular regulation in HCC. UBE4B is a U-box family of E4-active ubiquitin ligases that determine the specific recognition of substrates [16]. UBE4B can act as an oncogene or tumor suppressor gene in different types of cancers with opposite functions [13,25]. Zhang et al. showed that UBE4B influenced the development of HCC by promoting the growth of HCC cells, and silencing of UBE4B inhibited the proliferation, migration, and invasion of HCC cells, resulting in significant apoptosis [26]. However, a regulatory axis related to UBE4B in HCC is still lacking and requires detailed exploration.
In this study, the expression of UBE4B was analyzed in pan-cancer using TCGA and GTEx databases. UBE4B expression was found to be increased at both mRNA and protein levels in HCC compared with normal samples, and was associated with clinicopathological characteristics in the survival analysis. OS analysis also indicated the prognostic value of UBE4B in several cancers, including BLCA, LIHC, SARC, ESCA, HNSC, and KIRC. A previous study demonstrated that UBE4B expression is often upregulated at the transcriptional (71%) and translational (84%) levels in HCC tissues [16]. This report was consistent with our results, which illustrate the carcinogenic effects of UBE4B in HCC.
The correlation between UBE4B expression and HCC progression was investigated. UBE4B upstream miRNA expression patterns, more specifically hsa-miR-22-3p, also affected HCC in the starBase database. Recent studies have found that hsa-miR-22-3p acts as an important regulatory gene in breast cancer [27], Japanese encephalitis [28], and obesity [29]. The expression and prognosis of the upstream lncRNA of hsa-miR-22-3p, as a negative precursor molecule for miRNA, was analyzed using the starBase database, and five related lncRNAs (MIR4435-2HG, FGD5-AS1, LINC00858, SNAI3-AS1, and SNHG16) were shown to be significantly upregulated in HCC and associated with poor prognosis in patients with HCC. This is a topdown, molecular regulatory mechanism.
P53 protein, a well-known substrate of UBE4B, has been shown to regulate cell cycle arrest, apoptosis, and DNA repair processes, which play critical roles in preventing tumor progression [30,31]. This indicates that the overexpression of UBE4B leads to a carcinogenic process, with the amount of p53 protein greatly reduced in hepatocytes. Moreover, HCC cells have evolved to transfer oncogenic ncRNAs to recipient cells via exosomes, steadily transmitting biological signals and promoting tumor growth [32,33]. Similarly, it has been reported that miRNAs may also regulate the activity and function of p53 by directly targeting UBE4B [34]. However, there is no clear evidence for a relationship between hsa-miR-22-3p and tumorigenesis. Our results demonstrated a tumor process between UBE4B and HCC. Therefore, we propose a novel regulatory axis for targeted drugs, namely UBE4B-hsa-miR-22-3p-FGD5-AS1/LINC00858/SNHG16. Moreover, we confirmed that the mRNA expression of FGD5-AS1, LINC00858, SNHG16, and UBE4B was higher in HCC cell lines than that in normal hepatocyte lines, and the expression of hsa-miR-22-3p mRNA showed a decreasing trend.
Non-coding RNAs are important components of exosomes, and can regulate the TME and promote HCC progression, including development, progression, infiltration, metastasis, and angiogenesis [35]. These are traits of tumor immuno-infiltration and immune escape. Moreover, oncogenic ncRNAs from exosomes may also suppress the activity of immunosuppressive cells such as T-effector cells, ultimately leading to local or systemic immunosuppression. Our study showed the same mechanism, as evidenced by all the immune cells analyzed in HCC, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Furthermore, UBE4B expression was significantly and positively correlated with most immune cell biomarkers in HCC.
The degree of the immune response is regulated by the balance between the immunoinhibitor and immunostimulator pathways. These receptors and ligands are known as immune checkpoints [36]. Our study showed that UBE4B is significantly associated with immunoinhibitors and immunostimulators in HCC. The expression of UBE4B was significantly associated with many chemokines and chemokine receptors, suggesting that UBE4B may be associated with immune regulation in HCC and that these molecules could be potential immunotherapeutic targets for UBE4B in HCC. The current study had some limitations. First, most data are based on online databases that are constantly updated and expanded, which may affect the results of the study. Second, this study requires additional in vivo and in vitro experiments to examine the functional mechanisms of the ceRNA network. Third, these results need to be validated in large-scale clinical trials.

CONCLUSIONS
Taken together, our results suggest that UBE4B expression was increased in HCC and was correlated with a poor prognosis in patients with HCC. UBE4B expression was positively correlated with immune cell infiltration and immunomodulators, chemokines, and their receptors, further suggesting that UBE4B plays a carcinogenic role by modulating immune processes. Additionally, we identified the indispensable role of the UBE4B-hsa-miR-22-3p-FGD5-AS1/LINC00858/ SNHG16 regulatory axis in HCC. These findings will aid in understanding the relevant functions of UBE4B and provide new strategies for drug development and exploration of prognosis-related biomarkers.