A dataset for liver cancer was downloaded from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/). This study provides mRNA expression data for 372 tumors and 50 normal tissues, as well as clinical information such as age, clinical stage, gender, histological grade, TNM classification, survival time, survival status, and histological type. Data acquisition and applications were conducted in accordance with TCGA Publishing Guidelines and Data Access Policy. The local ethics committee does not need to approve this application.

Using R software (http://www.bioconductor.org/packages/release/bioc/html), we evaluated the differential expression of UBR5-related genes in liver cancer and normal samples. Pearson’s correlation analysis was used to identify gene-to-gene correlations. In the HCCDB database (http://lifeome.net/database), 15 public datasets are available, including data on UBR5 mRNA expression in liver cancer. According to the HCCDB database, liver cancer has a high expression of UBR5 compared to adjacent normal tissues. On the basis of the UALCAN website (http://ualcan.path.uab.edu/index.html), KM survival curves were analyzed to determine which genes affected survival. SPSS 17.0 (IBM, Chicago, IL, United States) and GraphPad Prism 6 (GraphPad Software, La Jolla, CA, United States) were used. UBR5 expressions have been compared between two groups using an unpaired t-test (two-tailed), with a 95% confidence interval, and a p value <0.05 deemed significant. In order to perform GO analysis, we used Database for Visualization, Annotation, and Integrated Discovery (DAVID) v6.7 (Leidos Biomedical Research, Inc., Bethesda, MD, United States).

Differentially expressed genes (DEGs) were identified. On the basis of the median score, the liver cancer samples were divided into two groups (high and low). R (version 4.0.3) and the R language package were used to test between high and low packets. For screening DEGs, FDR0.05 and |logFC|>1 were deemed statistically significant.

The PPI network connecting highly enriched DEGs was constructed using the string-db.org interacting gene search engine and displayed using Cytoscape. Prior to 19 DEGs, genetic screening interaction can have a maximum of 19 nodes. Proteins with a total score of interaction with UBR5 greater than 0.8 were chosen for further investigation.

Huh7 and Hep3B cells were obtained from the American Type Culture Collection (VA, United States). Cells were all cultured in Dulbecco’s modified medium (DMEM, Gibco, New York, United States), supplemented with 10% fetal bovine serum (FBS, Gibco, New York, United States), 100 μg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, United States), and 100 IU/ml penicillin G (Sigma-Aldrich) in a 5% CO₂, 37°C, and humidified incubator.

Liver cancer cells were seeded into 6-well or 24-well plates. After 24 h, the liver cancer cells were transfected with 20 nm UBR5 siRNA (GenePharma, Shanghai, China) or 20 nm NC siRNA (GenePharma, Shanghai, China). The siRNAs for UBR5 can be found in the Supplementary Table S1. In order to construct the YWHAZ plasmid, we inserted the full length of YWHAZ into the pcDNA3.1 vector. As instructed, Lipofectamine 3000 (Invitrogen, California, United States) was used to transfect related oligonucleotides and plasmids into liver cancer cells.

RIPA buffer (Thermo Scientific, Waltham, MA, United States) containing a protease inhibitor cocktail (Roche, Welwyn Garden, Switzerland, United Kingdom) was used to lyse tissue specimens and cells. Thirty micrograms of protein per sample were denatured in loading buffer.Proteins were separated on a 10% or 15% sodium dodecyl sulfate-polyacrylamide gel and transferred on to a nitrocellulose membrane (Bio-Rad, Hercules, United States). A 5% non-fat milk was applied, and the membrane was incubated overnight at 4°C with rabbit anti-UBR5 and rabbit anti-YWHAZ antibodies (1:1000; Santa Cruz, sc-515494 and 15222–1-AP respectively). After washing in phosphate buffered saline tween-20 (PBST), the membranes were incubated with horseradish peroxidase-goat anti-rabbit antibody (1:3000; Sigma-Aldrich, A0545) at room temperature for 2 h, then washed again in PBST. Western blots were visualized using enhanced chemiluminescence (ECL) detection reagent with an ECL kit (Cell Signaling Technology, Danvers, MA, United States). Antibodies are shown in Supplementary Table S2.

We used UBR5 and YWHAZ polyclonal antibodies at a dilution of 1:500 in immunohistochemistry assays on liver cancer tissue sections (UBR5: sc-9562, 1:500, Santa Cruz; YWHAZ: sc-518031, 1:100, Santa Cruz). The secondary antibody conjugated with horseradish peroxidase was applied at 37°C for 30 min, incubated with diaminobenzidine solution, and counterstained with hematoxylin.

Cell proliferation assay was performed using MTT following the manufacturer’s instructions. 1 × 10³ cells were plated in each well of a 96-well plate and incubated at 37°C for 24 h. We added 10 μl MTT (50 mg/ml) to each cell culture well. Then media was removed and 100 μl DMSO was added. The OD value was measured at an absorbance of 570 nm.

The expression of UBR5 in tumors and normal tissues from liver cancer patients was assessed using RNA-seq data from the TCGA database. UBR5 expression was significantly increased in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), cholangiocarcinoma (CHOL), liver hepatocellular carcinoma (LIHC), Glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (Figure 1). However, UBR5 expression was found in kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), uterine corpus endometrial carcinoma (UCEC), thyroid carcinoma (THCA), and thymoma (THYM) were substantially lower in cancer tissues than in non-carcinoma tissues. Based on these findings, UBR5 is implicated in the development of liver cancer, as previously found in other cancers.

Next, we investigated whether UBR5 expression was correlated to survival and clinicopathological features in liver cancer. The TCGA data showed that UBR5 was significantly expressed in liver cancer cohorts (Figure 2A). Furthermore, an investigation of 11 liver cancer groups in the HCCDB database revealed that UBR5 mRNA expression was considerably higher in 9 of the 11 groups including HCCDB1 (GSE22058), HCCDB3 (GSE25097), HCCDB4 (GSE36376), HCCDB6 [GSE14520(GPL3721 Subset)], HCCDB13 (GSE63898), HCCDB15 (TCGA-LIHC), HCCDB16 (GSE64041), HCCDB17 (GSE76427) and HCCDB18 (ICGC-LIRI-JP) when compared to adjacent tissues (Figure 2B, Supplementary Table S3). According to these findings, UBR5 expression is increased in liver cancer. Several clinicopathological characteristics of liver cancer patients were associated with UBR5 expression. Tumor grade (G4 vs. G1, p = 5.6E-03; G3 vs. G2, p = 5.6E−04; G3 vs. G1, p = 1.7E−06; G2 vs. G1, p = 4.7E−02), age (41–60 years vs. 61–80 years, p = 1.8E-02), weight (normal weight vs. obese, p = 9.1E-03), and TP53 mutation status (mutant vs. non-mutant, p = 9.1E-03) were strongly correlated with UBR5 expression (Figures 2C–J). Patients were divided into several survival subgroups according to the clinical characteristics of tumor grade, gender, Body Mass Index (BMI) and UBR5 expression. Compared to the low-risk group, the high-risk group had a lower OS rate in each subgroup (Figures 3A–D, Supplementary Figure S1). A negative correlation was observed between UBR5 expression levels and liver cancer prognosis in the study.

According to the findings presented above, UBR5 may play an important role in liver cancer. UBR5 coexpression genes were primarily involved in protein modification by small protein conjugation, cellular response to DNA damage stimulus, nuclear pore complex assembly, transcription by RNA polymeraseII, positive regulation of intracellular transport, nuclear pore organization, transcription by RNA polymerase II regulation, pore complex assembly, post-transcriptional gene silencing by RNA, and mitotic spindle organization (Figures 4A,B). According to KEGG pathway analysis (Figure 4C), these genes are primarily involved in ubiquitin-mediated proteolysis, RNA transport, basal transcription factors, and lysine degradation.

We used linkedomics (http://www.linkedomics.org/) to examine the coexpressed genes of UBR5 in 371 patients with liver cancer. Subsequently, significant DEGs in liver cancer were found to provide further understanding of the relationship between UBR5 expression and DEGs. TCGA provides an overview of the expression of UBR5-related genes in liver cancer through the construction of a gene expression heatmap. According to the heat map (Figures 5A,B), 24 genes were positively and negatively correlated with UBR5. A total of 48 genes (24 downregulated and 24 upregulated) were identified, 24 genes were significantly upregulated in liver cancer tumors compared to adjacent normal tissue (Figure 5A). Based on Pearson correlation analysis, Figure 5B shows that YWHAZ has a positive correlation with UBR5. A correlation coefficient of 0.83 was observed between YWHAZ and UBR5 (Figure 5C). APOC3 also showed a negative correlation with UBR5, and the correlation coefficient was −0.5 (Figure 5D). Using cytoscape software, a network of PPIs was constructed between significantly enriched DEGs. A maximum number of nodes can be found before 19 DEGs through the interaction between genetic screening and genetic mapping (Figure 5E). In the TCGA database, it was found that liver tumor groups displayed significantly higher levels of YWHAZ expression than normal groups.

Transient transfection experiments were used to knock down UBR5 expression in HCC cell lines. When compared to a negative control, UBR5 siRNA treatments reduced cell proliferation in Huh7 and Hep3B cells (Figures 6A–D). Transfecting a YWHAZ expression plasmid into UBR5-suppressed liver cancer cells restored YWHAZ expression. In a functional rescue experiment using Huh7 and Hep3B cells, YWHAZ clearly restored UBR5-mediated inhibition of proliferation (Figures 6C,D). By increasing YWHAZ expression, UBR5 promotes liver cancer cell growth.

In the TCGA data, YWHAZ was highly expressed in liver cancer cohorts (Figure 7A). Moreover Kaplan–Meier’s survival analysis revealed that patients with high levels of YWHAZ had shorter overall survival. Patients with high expressions of YWHAZ had significantly lower survival rates (Figure 7B). We found that our prognostic signatures were very robust at predicting OS for liver cancer patients.

Over the past decade, liver cancer diagnosis and treatment have improved significantly [14]. Despite widespread use of imaging in diagnosis and monitoring, liver cancer patients still have a low rate of early diagnosis. Currently, liver cancer is treated with surgical resection, chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy, and TACE [15–20]. Despite continuous advancements in comprehensive treatment technology, liver cancer suffers from a poor prognosis due to its high recurrence rate and lack of effective prognostic markers. The 5-year survival rate is less than 20%. It is imperative to identify potential biomarkers in liver cancer patients in order to improve their prognoses.

The UBR box E3 ligases are the major N-recognition proteins in the N-degron pathway [21, 22]. In addition to having a wide range of substrates, these N-recognition proteins play an important physiological role [23]. UBR5 has been shown to interact with a variety of cells directly in the process of various proteins, including cell cycle, transcription, and translation mechanisms, as well as DNA damage response and repair [24]. It is crucial to understand UBR5’s role in cancer development and progression. UBR5 has many molecular functions, and it has been linked to many aspects of cancer biology. The amplification of UBR5 is a common mutation in many cancers. For example, UBR5 amplification has been associated with increased UBR5 mRNA levels in breast cancer, primarily as a result of an allelic imbalance [25, 26]. It has been shown that UBR5 is involved in therapeutic resistance in ovarian cancer [9], possibly by regulating DNA damage response. Even though some results had been reported, the role of UBR5 phosphorylation remains unclear. UBR5 has thirty-four sites of phosphorylation reported in the literature; however, little is known about UBR5 phosphorylation and how it affects protein function. UBR5 has also been identified as an unfavorable prognosis marker in pancreatic cancer [27]. According to this study, UBR5 is up-regulated in hepatocellular carcinoma and acts as an oncogene. Bioinformatics analysis is required to assess the UBR5 regulation network comprehensively. These pathways may provide important therapeutic targets for future drugs if we can better understand how N-recognition proteins are mediated in them. It is generally known that UBR5 can be mutated in patients with liver cancer. Based on the TCGA database, liver cancer patients have a mutation rate of less than 2%. UBR5 is associated with tumor grade, age, weight, and TP53 mutation status in liver cancer. Poor prognosis is associated with a high level of UBR5 expression. The findings suggest that UBR5 may be involved in liver cancer diagnosis and prognosis.

UBR5 may play a role in liver cancer diagnosis and prognosis based on these findings. Although the Supplementary Figure S3 shows significantly increased expression of UBR5 and YWHAZ in the liver tumor group compared with the normal group from clinical samples, immunohistochemistry showed that UBR5 and YWHAZ were mainly localized in the nucleus, so these findings must be confirmed in clinical liver cancer patients as a further step.

The YWHAZ gene encodes the 14-3-3 protein, which participates in several signaling pathways by binding to ligands and plays an important role in tumor genesis and development [14]. Studies indicate that YWHAZ is frequently increased in malignancies such as hepatocellular carcinoma, colorectal cancer, lung cancer, and breast cancer [28–31]. A number of biological functions are performed by YWHAZ, including cell proliferation, apoptosis, migration, and invasion [31, 32].

UBR5, as a key oncogene, is progressively being demonstrated as a biomarker for liver cancer diagnosis and prognosis.