Overexpression pattern, function, and clinical value of proteasome 26S subunit non-ATPase 6 in hepatocellular carcinoma

Abstract

BACKGROUND

In recent years, many studies have shown that proteasome 26S subunit non-ATPase 6 (PSMD6) plays an important role in the occurrence and development of malignant tumours. Unfortunately, there are no reports on the evaluation of the potential role of PSMD6 in hepatocellular carcinoma (HCC).

AIM

To comprehensively evaluate the overexpression pattern and clinical significance of PSMD6 in HCC tissues.

METHODS

This study integrated PSMD6 mRNA expression profiles from 4672 HCC and 3667 non-HCC tissues, along with immunohistochemical scores from 383 HCC and adjacent tissues, to assess PSMD6 overexpression in HCC. Clustered regularly interspaced short palindromic repeats knockout technology evaluated PSMD6’s essential role in HCC cell growth. Functional enrichment analysis explored the molecular mechanism of PSMD6 abnormalities in HCC. Drug sensitivity analysis and molecular docking analysed the effect of abnormal expression of PSMD6 on the drug sensitivity of HCC cells.

RESULTS

The results of 41 external and two internal datasets showed that PSMD6 mRNA (SMD = 0.26, 95%CI: 0.09-0.42, P < 0.05) and protein (SMD = 2.85, 95%CI: 1.19-4.50, P < 0.05) were significantly overexpressed in HCC tissues. The integrated analysis results showed that PSMD6 had a significant overexpression pattern in HCC tissues (SMD = 0.40, 95%CI: 0.15-0.66, P < 0.05). PSMD6 knockout inhibited HCC cell growth (chronos scores < -1). Functional enrichment implicated ribosome biogenesis and RNA splicing. Significant enrichment of signalling pathways such as RNA degradation, ribosomes, and chemical carcinogenesis—reactive oxygen species. Drug sensitivity analysis and a molecular docking model showed that high expression of PSMD6 was associated with the tolerance of HCC cells to drugs such as ML323, sepantronium bromide, and GDC0810. Overexpressed PSMD6 effectively distinguished HCC tissues (AUC = 0.75, 95%CI: 0.71-0.79).

CONCLUSION

This study was the first to discover that PSMD6 was overexpressed in HCC tissues. PSMD6 is essential for the growth of HCC cells and may be involved in ribosome biogenesis and RNA splicing.

Key Words: Hepatocellular carcinoma; Proteasome 26S subunit non-ATPase 6; Clustered regularly interspaced short palindromic repeats; Ribosome biogenesis; RNA splicing

Core Tip: This study integrated 8339 hepatocellular carcinoma (HCC) tissue mRNA expression profiles from 41 platforms, 383 pairs of hepatocellular tissue samples from inhouse immunohistochemistry experiments, and HCC cell lines using the clustered regularly interspaced short palindromic repeats system for proteasome 26s subunit non-ATPase 6 (PSMD6) knockout data to evaluate the abnormal expression pattern and clinical significance of PSMD6 in HCC tissues. This study was the first to discover that PSMD6 was overexpressed in HCC tissues. The promotion of PSMD6 in HCC progression is closely related to ribosome biogenesis and RNA splicing, suggesting that PSMD6 may enhance the proliferation of HCC cells.

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common malignancies and is strongly linked with several key risk factors: Hepatitis virus infection, aflatoxin exposure, metabolic disorders, heavy alcohol consumption and smoking[1]. The high infection rates of hepatitis B virus, hepatitis C virus (HCV), and aflatoxin are important reasons for the long-term high morbidity and mortality of HCC[2-5]. Due to the lack of clinical symptoms in the early stages of HCC, many patients are diagnosed in the late stages[6]. For early HCC, common treatments are liver transplantation and surgical resection; for advanced HCC, common treatments are radiofrequency ablation, transarterial chemoembolization, targeted therapy, and immunotherapy[6]. Although there are many treatment options that have had a positive impact on patients with advanced HCC, drug resistance and recurrence still exist[7]. Timeliness and accuracy of diagnosis are still the keys to improving the prognosis of HCC[8]. Therefore, further understanding of the molecular biological mechanisms related to HCC is crucial to improving the timeliness of HCC diagnosis, treatment efficacy, and patient prognosis.

Proteasome 26S subunit non-ATPase 6 (PSMD6) is one of the important components of the ubiquitin-proteasome system. In recent years, many studies have shown that PSMD6 plays an important role in the occurrence and development of malignant tumours. Some studies have reported that overexpression of PSMD6 in pancreatic cancer is significantly associated with poor overall survival[9]. The prognostic model of oesophageal cancer involving PSMD6 has a high accuracy in predicting prognosis and judging the sensitivity of targeted therapy[10]. PSMC6, which has a highly correlated expression pattern with PSMD6, has the effect of promoting the growth and metastasis of lung adenocarcinoma cells[11]. Unfortunately, however, there are no reports on the evaluation of the potential role of PSMD6 in HCC.

To this end, this study is the first to comprehensively evaluate the abnormal expression pattern, potential molecular mechanism, and clinical significance of PSMD6 in HCC tissues based on multicentre large-sample bulk RNA-Seq, inhouse immunohistochemistry, and clustered regularly interspaced short palindromic repeats (CRISPR) knockout screen technology.

MATERIALS AND METHODS

In-house immunohistochemistry to evaluate the expression pattern of PSMD6 protein in HCC tissues

In this study, 284 and 99 HCC tissues and their corresponding adjacent tissues were collected from The First Affiliated Hospital of Guangxi Medical University (hereinafter referred to as Institution A) and Redcross Hospital of Yulin (hereinafter referred to as Institution B), respectively. 16 and 5 tissue microarrays were constructed from 284 and 99 pairs of tissue samples, respectively. PSMD6 antibody [rabbit polyclonal antibody (AB155761)] was purchased from Abcam Plc (Shanghai, China). All technical operations on tissue microarray and immunohistochemistry were strictly performed in accordance with the manufacturer's technical standards. Immunohistochemical scoring was performed independently by two senior pathologists. Positive cell counts were by randomly examining 100 cells in each field of view under the highest setting of the available equipment. If the number of positive cells is 0-5, 6-15, 16-50, 51-75, and greater than 75, the positive cell score of the field of view is 0, 1, 2, 3, and 4 points, respectively. Staining intensity is scored on a scale of 0 to 3: 0 no staining, 1 indicates weak staining, 2 signifies moderate staining, and 3 denotes strong staining. Ten fields of view are randomly selected for analysis in each tissue sample. The product of the positive cell score and the staining intensity score in a field of view is the immunohistochemical score of the field of view. The average of the immunohistochemical scores of 10 fields of view in a sample is the final score of the sample. The final immunohistochemical score reflects the expression level of PSMD6 protein in the tissue sample. The in-house immunohistochemical experiment of this study was approved by the Ethics Committee of Institution A (No. 2023-E511-01) and the Ethics Committee of Institution B (No. 2024 Ethics-8). All patients or their families involved gave informed consent.

Collection, organisation, and analysis of PSMD6 mRNA expression profiles in HCC tissues

This study widely collected bulk RNA-Seq data and corresponding clinical data of CRC tissues available worldwide from databases such as the Cancer Genome Atlas (TCGA), Gene Expression Omnibus, Sequence Read Archive, ArrayExpress, and International Cancer Genome Consortium. The specific process of retrieving and screening datasets is detailed in Figure 1. We finally included PSMD6 mRNA expression profiles of 4672 HCC tissues and 3667 non-HCC tissues from 41 platforms. Then, we merged, de-batched, standardised, corrected, and removed abnormal data samples based on platform information. The standardised mean difference (SMD) of PSMD6 was calculated to evaluate the abnormal expression pattern of PSMD6 mRNA in HCC tissues. External mRNA datasets and internal immunohistochemistry datasets were integrated and analysed to further verify the abnormal expression of PSMD6 in HCC tissues. In addition, SMD sensitivity was used to evaluate the stability of SMD. Begg’s test and Egger’s test were used to detect publication bias.

Figure 1 Flowchart for the collection of proteasome 26S subunit non-ATPase 6 bulk RNA-Sequencing datasets from hepatocellular carcinoma tissues. HCC: Hepatocellular carcinoma; TCGA: The Cancer Genome Atlas; GTEx: Genotype-Tissue Expression; GEO: Gene Expression Omnibus; ICGA: International Cancer Genome Consortium; PSMD6: Proteasome 26S subunit non-ATPase 6.

Evaluation of the effect of PSMD6 on the growth status of HCC cell lines

This study used CRISPR knockout screen technology from the DepMap database to analyse the effect of PSMD6 on the growth of 20 primary HCC cell lines[12]. After the PSMD6 gene was knocked out in the HCC cell lines, the chronos algorithm was used to evaluate the PSMD6 chronos score to evaluate the effect of PSMD6 knockout on the growth of HCC cell lines[13]. In this study, a chronos score less than -1 indicated that PSMD6 was highly necessary for the growth and proliferation of the cell line.

Functional enrichment analysis

Based on the included multi-centre mRNA expression profiles, this study used the meta and metafor packages of R v4.2.2 to batch calculate the SMD of each gene to screen out differentially expressed genes (DEGs) (the 95%CI of SMD does not include 0, P < 0.05). Next, we used cBioPortal to analyse the HCC mRNA data from TCGA to identify genes co-expressed with PSMD6 (|R| > 0.25, P < 0.05)[14]. By taking the intersection of DEGs and PSMD6 co-expressed genes, we obtained the intersection genes. Subsequently, we performed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on these intersection genes to explore the possible molecular biological mechanism of PSMD6 in HCC.

Drug sensitivity analysis of HCC

Based on the HCC tissue mRNA expression profile in the TCGA database and the GDSC 2.0 database, the correlation between PSMD6 mRNA expression level and targeted drug sensitivity score (as measured by IC₅₀ value) was calculated using the oncopdict package R v 4.2.2. The effect of PSMD6 expression on IC₅₀ was evaluated by regression analysis. In this study, if there is a strong correlation between PSMD6 mRNA expression level and drug sensitivity (|R|> 0.35, P < 0.05), it is believed that PSMD6 has a significant effect on the drug sensitivity of targeted therapeutic drugs in HCC cells.

Construction of molecular docking model between PSMD6 protein and drugs

This study obtained the three-dimensional structure of PSMD6 protein (PDB format) from the PDB database and the three-dimensional structure (sdf format) of small molecule targeted therapeutic drugs from PubChem. This study also used CB-Dock2 to repair missing atoms and hydrogen atoms, remove water molecules and other heteroatoms, detect protein pockets, estimate spatial parameters, and perform Vina scoring on the three-dimensional structure of PSMD6 protein and small molecule targeted therapeutic drugs for protein drug blind docking analysis[15]. The molecular docking model with the best Vina score is visually displayed. In this study, Vina scores less than -6 kcal/mol are considered to have good binding ability; scores greater than -6 and less than -4 kcal/mol are considered to have average binding ability; scores greater than -4 kcal/mol are considered to have poor binding ability.

Assessment of the clinical significance of abnormal PSMD6 expression patterns in HCC tissues

This study used receiver operating characteristic curve (ROC), summary ROC (sROC), positive likelihood ratio, and negative likelihood ratio forest plots to analyse the above-mentioned collected HCC PSMD6 mRNA expression profiles, HCC tissue internal protein expression levels, and corresponding clinical information. The ability of PSMD6 expression levels to identify HCC tissues was evaluated. Since most of the HCC patients in Institution B's internal samples have relatively complete clinical information, this study also evaluated the relationship between PSMD6 protein expression levels and the clinicopathological characteristics of corresponding HCC patients to further analyse the influencing factors of PSMD6 expression levels.

Statistical analysis

All statistical analyses involved in this study were completed on R v4.2.2, Stata v12, and SPSS v23. Q test and I² statistical analysis were used to select the calculation model to be used to calculate the SMD of PSMD6. If I² > 50%, it indicates that there is large heterogeneity, and the random effects model is used to calculate SMD. Otherwise, the fixed effects model was used. When comparing the differences in the means of two or more groups, choose the t test, one-way analysis of variance, or rank sum test according to the type and characteristics of the data. In this study, P < 0.05 was considered statistically significant.

RESULTS

Overexpression pattern of PSMD6 protein in HCC tissues

In the study, 383 HCC tissues and their corresponding paracancerous tissues were collected for in-house immunohistochemistry to evaluate the overexpression pattern of PSMD6 protein in HCC tissues. From a qualitative perspective, PSMD6 showed positive or strong positive staining in HCC tissues (Figure 2A and B). PSMD6 showed weak positive or negative staining in the corresponding paracancerous tissues of HCC (Figure 2C and D). To more objectively evaluate the overexpression pattern of PSMD6, we also scored the inhouse immunohistochemistry results for quantitative analysis. The results showed that whether in institution A samples (Figure 3A) or in institution B samples (Figure 3B), the PSMD6 protein expression level in HCC tissues was significantly higher (P < 0.05) than the corresponding paracancerous tissues.

Figure 2 Qualitative evaluation of proteasome 26S subunit non-ATPase 6 protein expression in hepatocellular carcinoma and non-hepatocellular carcinoma tissues. A and B: Hepatocellular carcinoma (HCC) tissue; C and D: Non-HCC tissue.

Figure 3 Semi-quantitative evaluation of proteasome 26S subunit non-ATPase 6 protein expression in hepatocellular carcinoma and non-hepatocellular carcinoma tissues. A and B: Semi-quantitative comparison of proteasome 26S subunit non-ATPase 6 (PSMD6) protein expression levels between HCC tissues and non-HCC tissues from institutions; C and D: Evaluation of the ability of highly expressed PSMD6 from institutions A and B to identify HCC tissues. HCC: Hepatocellular carcinoma; PSMD6: Proteasome 26S subunit non-ATPase 6.

PSMD6 is significantly highly expressed in HCC tissues

This approach aims to the findings of high PSMD6 in HCC tissues by accessing and analyzing a large, diverse dataset of RNA data from multiple sources. Since I² is all greater than 50%, the random effects model is used to calculate PSMD6 SMD (Figure 4). Results from 41 external datasets and 2 internal datasets showed that PSMD6 mRNA (SMD = 0.26, 95%CI: 0.09-0.42, P < 0.05) and protein (SMD = 2.85, 95%CI: 1.19-4.50, P < 0.05) are significantly highly expressed in HCC tissues (Figure 4). The integration of external and internal data further showed that PSMD6 has a significant overexpression pattern in HCC tissues (SMD = 0.40, 95%CI: 0.15-0.66, P < 0.05; Figure 4). Sensitivity analysis of PSMD6 SMD showed that no significant changes were found when PSMD6 SMD was re-evaluated by discarding individual HCC tissue data sets one by one (Figure 5A). In addition, Begg’s test (P > 0.05) and Egger’s test (P > 0.05) did not detect significant publication bias (Figure 5B). These results illustrate that external and internal datasets from multiple centres can be combined for analysis without appreciable impact on the results.

Figure 4 Proteasome 26S subunit non-ATPase 6 mRNA and protein have significant overexpression patterns in hepatocellular carcinoma tissues. SMD: Standardised mean difference.

Figure 5 Proteasome 26S subunit non-ATPase 6 standardised mean difference reliability test. A: Forest plot of sensitivity analysis of proteasome 26S subunit non-ATPase 6 (PSMD6) standardised mean difference (SMD); B: Funnel plot of publication bias test of PSMD6 SMD. SMD: Standardised mean difference.

PSMD6 is necessary for the growth of HCC cells

This study used CRISPR knockout screen technology to analyse the effect of PSMD6 on the growth of 20 HCC cell lines. The gene chronos score of PSMD6 in 20 types of HCC cells showed that after knocking out the PSMD6 gene, all 20 types of HCC cells showed significant growth inhibition (chronos scores were all less than -1; Figure 6). This result suggests that the PSMD6 gene is an essential gene for HCC cell growth.

Figure 6 Gene knockout effect score of proteasome 26S subunit non-ATPase 6 in hepatocellular carcinoma cell lines. PSMD6: Proteasome 26S subunit non-ATPase 6.

Potential biological mechanisms of PSMD6 in HCC

This study obtained 600 intersection genes through differential analysis and co-expression analysis. GO enrichment analysis showed that PSMD6 abnormally expressed in HCC might influence or disrupt signalling pathways such as the tRNA metabolic process, ncRNA processing, ribosome biogenesis, RNA splicing, and RNA localization to the nucleus. In addition, PSMD6 is involved in the biogenesis of ribosomes, suggesting that it may contribute to dysregulation of protein synthesis. Its effect on RNA splicing may lead to the production of oncogene subtypes that drive HCC progression. Alterations in ncRNA processing may also lead to dysregulation of tumor suppressor rna or accumulation of carcinogenic mirnas (Figure 7A). KEGG enrichment analysis showed significant enrichment of signalling pathways such as RNA degradation, ribosomes, and chemical carcinogenesis—reactive oxygen species (Figure 7B). These molecular pathways highlight the potential role of PSMD6 as a key regulator in the development and progression of HCC.

Figure 7 Evaluation of the potential molecular biological mechanisms of proteasome 26S subunit non-ATPase 6 in hepatocellular carcinoma tissues. A: Gene Ontology enrichment analysis; B: Kyoto Encyclopedia of Genes and Genomes enrichment analysis.

Effect of PSMD6 on drug sensitivity of HCC cells

Drug sensitivity analysis showed that the drug sensitivity of ML323 (R = -0.40, P < 0.05), Sepantronium bromide (R = -0.35, P < 0.05), GDC0810 (R = -0.35, P < 0.05), BPD-00008900 (R = -0.35, P < 0.05), and Bortezomib (R = -0.35, P < 0.05) in HCC cells was negatively correlated with the expression level of PSMD6 mRNA (Figure 8). This suggests that HCC cells overexpressing PSMD6 have a certain tolerance to these targeted therapeutic drugs. In addition, this study also constructed a molecular docking model to further evaluate this drug-sensitivity relationship. Given that BPD-00008900 and Bortezomib do not have 3D structures suitable for this analysis process, only the docking models of the other three small molecules with PSMD6 are shown here. The results showed that ML323 (Vina score = -7.5 kcal/mol), Sepantronium bromide (Vina score = -6.1 kcal/mol), and GDC0810 (Vina score = -6.6 kcal/mol) all had strong binding ability with PSMD6 protein (Figure 9). And there were strong intermolecular forces in these three molecular docking models (Figure 9). For example, hydrogen bond, Pi-Pi stacking, and hydrophobic interaction. This further illustrates the stability of these molecular docking models.

Figure 8 Correlation analysis between drug sensitivity of tumour targeted therapy drugs and proteasome 26S subunit non-ATPase 6 mRNA expression level in hepatocellular carcinoma tissues. PSMD6: Proteasome 26S subunit non-ATPase 6.

Figure 9 Construction of molecular docking model between proteasome 26S subunit non-ATPase 6 protein and small molecule compounds. A: Sepantronium bromide; B: ML323; C: GDC0810.

Clinical and pathological significance of an abnormal expression pattern of PSMD6 in HCC tissue

First, in the immunohistochemical samples of institution A (AUC = 0.972; Figure 3C) and institution B (AUC = 0.897; Figure 3D), the highly expressed PSMD6 protein had a strong recognition ability for HCC tissue. The combination of external multicentre PSMD6 mRNA and internal protein analysis further demonstrated that PSMD6 had a strong ability to discriminate HCC tissues (AUC = 0.75, 95%CI: 0.71-0.79; Sensitivity = 0.60, 95%CI: 0.49-0.71; Specificity = 0.79, 95%CI: 0.68-0.87; Positive likelihood ratio = 2.83, 95%CI: 1.93-4.14; Negative likelihood ratio= 0.50, 95%CI 0.39-0.65; OR = 5.62, 95%CI: 3.34-9.43; Figure 10 and Figure 11). In addition, Table 1 suggests that the expression level of PSMD6 in HCC tissues of HCV-positive patients was higher than that of HCV-negative patients (P < 0.05). The remaining clinicopathological characteristics (e.g., age, gender, vascular invasion, tumour necrosis, hepatitis grade, satellite nodules, and Alpha-fetoprotein) were not statistically significant (P > 0.05).

Figure 10  Sensitivity and specificity analysis of proteasome 26S subunit non-ATPase 6 mRNA and protein in identifying hepatocellular carcinoma tissues. A: Summary receiver operating characteristic curve; B: Forest plot of sensitivity and specificity. ROC: Receiver operating characteristic curve; sROC: Summary receiver operating characteristic curve.

Figure 11  Evaluation of the ability of proteasome 26S subunit non-ATPase 6 mRNA and protein expression levels to identify hepatocellular carcinoma tissues. A: Forest plot of likelihood ratio; B: Forest plot of diagnostic score and odds ratio.

Table 1 Association between clinicopathological information and proteasome 26S subunit non-ATPase 6 protein expression levels in inhouse hepatocellular carcinoma tissue samples.

Clinicopathological Features		PSMD6 protein expression
		n	Mean	SD	t (t-test) or F (ANOVA)	P value
Gender	Male	82	9.06	2.94	-0.93	0.36
	Female	17	9.79	2.91
Age	> 60 years	28	8.77	2.99	-0.89	0.38
	≤ 60 years	71	9.35	2.92
Tumour necrosis	Yes	34	9.08	2.88	-0.26	0.80
	No	65	9.24	2.99
Tumour recurrence	Yes	4	9.00	3.60	-0.13	0.90
	No	95	9.20	2.93
Number of lesions	1	91	9.20	2.88	0.09	0.93
	> 1	8	9.10	3.73
Satellite nodules	Yes	9	9.29	2.04	0.11	0.92
	No	90	9.18	3.02
Vascular invasion	M0	50	8.79	3.07	1.34	0.27
	M1	35	9.38	2.82
	M2	13	10.22	2.60
Lymph node invasion	Yes	10	9.88	1.92	1.12	0.29
	No	89	9.11	3.03
Hepatitis grade	G0	6	8.07	3.49	0.58	0.63
	G1	36	8.93	2.86
	G2	54	9.44	3.00
	G3	3	10.00	1.74
Liver fibrosis grading	S1	9	9.87	2.71	0.23	0.88
	S2	19	8.88	3.72
	S3	12	9.03	3.41
	S4	52	9.15	2.59
T stage	T1	45	8.75	3.04	1.10	0.34
	T2	44	9.65	2.87
	T3	9	8.84	2.69
N stage	N0	96	9.18	2.97	-0.09	0.93
	N1	3	9.33	2.31
HBV	Positive	86	9.02	2.96	-1.44	0.15
	Negative	13	10.28	2.65
HCV	Positive	5	6.32	3.65	-2.29	0.02
	Negative	94	9.34	2.84
AFP	> 400 ng/mL	41	9.35	2.91	0.45	0.65
	< 400 ng/mL	58	9.08	2.98
BCLC staging	A	81	9.27	2.93	0.15	0.86
	B	13	8.80	3.04
	C	3	9.33	2.31

HBV: Hepatitis B virus; HCV: Hepatitis C virus; PSMD6: Proteasome 26S subunit non-ATPase 6; AFP: Alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer.

DISCUSSION

Currently, HCC patients have not only a large base number but also a very poor prognosis. HCC accounts for 90% of liver cancer cases[16]. In 2022, the numbers of new cases of and deaths from liver cancer worldwide were 865269 and 757948, respectively, accounting for 4.3% and 7.8% of the total number of new cancer cases and total tumour-related deaths, respectively[17]. The five-year survival rate of HCC is less than 20%[16,18]. This situation is closely related to the ongoing challenges in achieving an early diagnosis of HCC[18]. Therefore, further research on molecular biomarkers related to HCC remains of great significance. At present, no studies have reported the abnormal expression pattern, molecular biological behaviour, and clinical pathological significance of PSMD6 in HCC. Consequently, this study integrated multiple pieces of evidence for the first time, including mRNA expression profiles of HCC tissues, in-house immunohistochemistry experiments on HCC tissues and corresponding paracancerous tissues, a CRISPR knockout screen of PSMD6 in 20 HCC cell lines, drug sensitivity analysis, and the construction of a molecular docking model, to comprehensively evaluate the overexpression pattern and clinical significance of PSMD6 in HCC tissues.

Immunohistochemistry can intuitively evaluate the expression and spatial distribution characteristics of target proteins at the histological level, which sheds light on the potential role of target proteins in the occurrence and development of tumours[19]. In this study, a total of 383 pairs of HCC tissues and corresponding paracancerous tissues was collected from institutions A and B for in-house immunohistochemistry experiments, and the results showed that PSMD6 was significantly overexpressed in HCC tissues compared with paracancerous tissues. Given that results based on single-centre datasets can lack reliability, this study also collected 8,339 Liver tissue mRNA expression profiles from multiple external dataset libraries. This study then integrated and analysed these 41 external datasets and two internal datasets, and it was concluded that PSMD6 has an overexpression pattern in HCC tissues. Therefore, the results of this study, which was based on the design of a large external multicentre sample and internal sample experiments, have strong generalisability and reliability. In addition, the results of the CRISPR knockout screen technology analysis showed that the growth of HCC cells is dependent on PSMD6. Therefore, abnormal PSMD6 may play a certain role in the occurrence and development of HCC.

This study therefore conducted functional enrichment analysis to further explore the potential molecular biological behaviour of PSMD6 in HCC tissues. The above results show that the overexpression pattern of PSMD6 in HCC involves abnormal pathways, such as ribosome biogenesis and RNA splicing. Although this study is the first to reveal the connection between PSMD6 in HCC and these pathways, some previous related studies support our results. Under the action of some inducers, the overactivation of ribosome biogenesis and the imbalance of related proteins can promote the occurrence and development of HCC[20]. Other studies have pointed out that the upregulation of ribosome biogenesis and mRNA translation efficiency can promote the progression of HCC by promoting the self-renewal of liver cancer stem cells[21]. A series of in-vitro and in-vivo experiments has shown that ovatodiolide and antrocin can synergistically inhibit the stemness and metastatic potential of HCC cells by downregulating ribosome biogenesis[22]. In addition, in tumour cells, the expression level of the ribosome biogenesis factor WBSCR22 is regulated by the 26S proteasome, which is composed of the PSMD6 protein[23]. Moreover, in terms of RNA splicing, abnormal RNA splicing can promote the progression of HCC by upregulating TAK1-MAPK signal transduction[24]. At the same time, RNA splicing can enhance the stemness and proliferation activity of HCC cells[25]. IK (a spliceosome component) regulated by the 26S proteasome is a key factor in correct mRNA splicing[26]. Therefore, it is reasonable that PSMD6 plays a role in promoting the progression of HCC by participating in ribosome biogenesis and RNA splicing pathways. These results guide us to focus on the relationship between PSMD6, ribosome biogenesis, RNA splicing, and HCC and have important implications for our future research.

In addition, given the clinical potential of the overexpression pattern of PSMD6 in HCC tissues, this study evaluated the drug sensitivity and clinical pathological significance of targeted therapeutic drugs. In terms of drug sensitivity, ML323, sepantronium bromide, GDC0810, BPD-00008900, and bortezomib have great potential anti-HCC effects. Previous studies have shown that bortezomib can inhibit the 26S proteasome in which PSMD6 protein participates and thus reduce the growth of HCC cells[27], confirming PSMD6 as a potential therapeutic target. In-vivo experiments have shown that ML323 can exert its anti-HCC function by inhibiting USP1[28], and targeting PSMD6 may increase the sensitivity of HCC cells to ML323 and similar drugs. Sepantronium bromide (YM155) can inhibit the proliferation of HCC cells by acting on genes related to cell cycle checkpoints[29]. HCC with a high m5C score is more sensitive to the effects of sepantronium bromide[30]. The latest study shows that GDC0810 has a better therapeutic effect on HCC with high-risk disulfidptosis-related prognostic model scores[31]. Other studies have shown that BPD-00008900 and ML323 have better therapeutic effects on HCC patients with high cholesterol metabolism[32], further illustrating the complex interaction between metabolic pathways and drug sensitivity. In this study, we revealed a negative correlation between the sensitivity of these drugs and the expression level of PSMD6. Therefore, PSMD6 is an important factor affecting sensitivity and resistance to these drugs. Future studies should focus on the exact mechanism by which PSMD6 affects drug reactivity, thereby potentially enabling innovative combination therapies to overcome resistance and improve patient outcomes. In terms of clinical pathological significance, the results of sROC, ROC, and positive likelihood ratio and negative likelihood ratio forest plots showed that the overexpression pattern of PSMD6 mRNA and protein has a high clinical potential for the early diagnosis of HCC. However, as these results are mainly based on histological studies, there are certain limitations on their clinical translation value. In the future, it is necessary to explore whether the PSMD6 expression status in HCC body fluids that can be obtained by non-invasive technology has a similar overexpression pattern as HCC tissue and to evaluate its feasibility for the early diagnosis of HCC. Nevertheless, these research results provide a certain theoretical basis for the early diagnosis of HCC. In addition, this study revealed for the first time that the expression level of PSMD6 in the HCC tissues of HCV-positive patients was higher. However, this result may be biassed by the small number of HCV-positive HCC patients in this study. In the future, it will be necessary to expand the sample for further research.

Although this study makes many new findings, the following limitations are worth noting. First, the clinical information of some in-house immunohistochemistry samples in this study was not fully collected; for example, all the in-house samples lack prognosis and follow-up information. In the future, we will improve the way information is collected to obtain more valuable results. Second, although this study explored the abnormal molecular biological behaviour of PSMD6 in HCC for the first time based on a large multicentre sample dataset, in-depth future research is needed to verify and supplement the results. In addition, the results of the drug sensitivity analysis and molecular docking models revealing the effect of PSMD6 on the drug sensitivity of some targeted therapeutic drugs are based on in-vitro cell line experiments and computer simulations. Therefore, in the future, we need to carry out more in-vivo experiments, as well as clinical translational research, and apply the predicted results in vitro to clinical trials to verify their effect in actual treatment.

CONCLUSION

This study used the mRNA expression profile from external multicentre and inhouse immunohistochemistry experiments and found for the first time that PSMD6 has an overexpression pattern in HCC tissues. PSMD6 is very necessary for the growth of HCC cells. This growth necessity may be related to pathways such as ribosome biogenesis and RNA splicing. They hold promise for improving the prognosis of HCC through the targeting of PSMD6.

ACKNOWLEDGEMENTS

We thank the Guangxi Zhuang Autonomous Region Clinical Medicine Research Center for Molecular Pathology and Intelligent Pathology Precision Diagnosis for providing technical support in computational pathology and experimental pathology.