Overexpression of proteasome 26S subunit non-ATPase 6 protein and its clinicopathological significance in intrahepatic cholangiocarcinoma

Abstract

BACKGROUND

Currently, intrahepatic cholangiocarcinoma (ICC) poses a continuing, significant health challenge, but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6 (PSMD6).

AIM

To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.

METHODS

The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology. Forty-two paired specimens of ICC and adjacent non-cancerous tissues were collected. PSMD6 protein expression was determined by immunohistochemistry. Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level, and its association with ICC patients’ various clinicopathological characteristics was investigated.

RESULTS

The PSMD6 gene was found to be essential for the growth of ICC cell lines. PSMD6 protein was significantly overexpressed in ICC tissues (P < 0.001), but showed no significant association with patient age, gender, pathological grade, or tumor-node-metastasis stage (P > 0.05).

CONCLUSION

PSMD6 can promote the growth of ICC cells, thus playing a pro-oncogenic role.

Key Words: Intrahepatic cholangiocarcinoma; Proteasome 26S subunit non-ATPase 6; Immunohistochemistry; Clustered regularly interspaced short palindromic repeat knockout screening; Clinicopathological characteristics

Core Tip: This study examined the expression and clinicopathological significance of proteasome 26S subunit non-ATPase 6 (PSMD6) in intrahepatic cholangiocarcinoma (ICC). It was discovered that the PSMD6 gene was essential for the proliferation of ICC cell lines. Furthermore, PSMD6 protein expression was significantly elevated in ICC specimens (P < 0.001), although it was not significantly correlated with patient age, gender, pathological grade, or tumor-node-metastasis stage (P > 0.05). These results indicate that PSMD6 may enhance the proliferation of ICC cells, thereby contributing to its oncogenic potential.

INTRODUCTION

Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor and the second most common primary liver cancer. ICC accounts for 10%-20% of primary liver cancer cases[1-4] and its incidence is increasing worldwide[5-8]. Therefore, identifying molecular biomarkers related to ICC is crucial to enhancing the accuracy of early diagnosis, the effectiveness of treatment, and patient prognosis.

The proteasome 26S subunit non-ATPase 6 (PSMD6) gene, located on chromosome 3p14.1, consists of 11 exons and encodes a subunit of the 26S proteasome, which is a member of the ubiquitin-proteasome system. The ubiquitin-proteasome system is responsible for recognising and degrading proteins and plays a crucial role in protein quality control, cell cycle regulation, gene expression, and lifespan regulation[9]. It is also involved in various biological processes with relevance to diseases such as tumours and diabetes[10,11], yet there have been no reports on the association between PSMD6 and ICC. Therefore, this study used immunohistochemistry (IHC) to investigate the expression of PSMD6 protein in ICC tissues and explore its correlation with various clinicalpathological characteristics.

MATERIALS AND METHODS

Potential impact of PSMD6 gene on ICC cells

This study employed clustered regularly interspaced short palindromic repeat (CRISPR) knockout screening technology to elucidate the effect of the PSMD6 gene on ICC cell growth. ICC cell lines were subjected to PSMD6 gene knockout, and the CERES algorithm was used to calculate the PSMD6 gene effect scores to evaluate the knockout effects of PSMD6 in ICC cell lines. A negative score indicated inhibited proliferation of ICC cells after PSMD6 gene knockout[12]. In this study, if more than 75% of ICC cell lines had a score less than -1, the gene was deemed as a growth-associated gene for ICC[13].

Expression of PSMD6 protein in ICC tissues

This study used The Human Protein Atlas (THPA) database (https://www.proteinatlas.org/) and in-house tissue microarrays to explore the PSMD6 protein expression in ICC tissues. We obtained immunohistochemical images of ICC tissue and normal bile duct epithelial tissue from the THPA database[14]. Tissue microarrays (LVC1261) containing 42 pairs of ICC specimens and corresponding adjacent non-cancerous tissues were obtained from Pantomics, Inc. None of the tissue donor patients received any preoperative therapeutic interventions, and cholangiocarcinoma was the pathological type of all specimens. The distance between the ICC tissues and adjacent non-cancerous tissues was at least 3 cm. Relevant clinicopathological characteristics were collected, including age, gender, pathological grade, and tumor-node-metastasis (TNM) stage. The Pantomics, Inc. ethics committee granted ethical approval for this study [Ethics No. Fanpu(2018)23].

Tissue sections of 2 μm thickness were prepared from paraffin-embedded tissue blocks using a microtome. These sections were subsequently incubated at 75 °C for 30 min to remove the wax. After dewaxing, the sections were treated with 3% H₂O₂ for 15 min, followed by rinsing with distilled water and phosphate-buffered saline (PBS). The sections were then incubated with a polyclonal rabbit anti-PSMD6 antibody (Abcam, catalog No. ab155761) and subsequently washed with PBS. The tissue microarrays were subjected to restaining, dehydration, transparency, and sealing processes. Adjacent tissue sections served as the positive control, whereas PBS alone was used as the negative control.

This study integrated data from the THPA database with the IHC results to summarise and analyse the expression of PSMD6 in ICC tissue and adjacent tissue samples. Two blinded, experienced pathologists independently evaluated the slides. The results were assessed by the proportion of positive cells and staining intensity. The percentage of positive cells was scored as 0, 1, 2, 3, and 4 points, respectively, for no expression, < 10%, 10%-35%, 36%-75%, and > 75%. Staining intensity was scored as 0, 1, 2, and 3 points, respectively, for no or almost no cytoplasmic staining, light yellow, and brown yellow[15-17]. The final score for each specimen was calculated by multiplying the percentage score by the intensity score. If the final scores of two pathologists were inconsistent, the average score was taken.

Statistical analysis

Data processing was conducted using SPSS 19.0 software. The association between PSMD6 protein expression and patients’ clinicalpathological characteristics was analyzed using the χ² test and Spearman correlation analysis. The expression differences of the PSMD6 protein in ICC tissues and adjacent non-cancerous tissues were compared using the t-test. A violin plot and receiver operating characteristic (ROC) curve were generated using the ggplot2 package in R (v4.3.1). All results were subjected to two-tailed tests, with statistical significance set at P < 0.05.

RESULTS

Growth status of ICC cells after PSMD6 gene knockout

This study used the CRISPR knockout screening technology to assess the PSMD6 gene’s impact on the proliferation of ICC cells. The gene effect scores of PSMD6 in 17 ICC cell lines indicate significant growth inhibition in all 17 ICC cell lines after PSMD6 gene knockout (Figure 1). The gene effect scores were all less than -1, suggesting that the PSMD6 gene is a growth-associated gene for ICC cells (17/17, 100%).

Figure 1 Growth inhibition of 17 intrahepatic cholangiocarcinoma cell lines after proteasome 26S subunit non-ATPase 6 gene knockout. PSMD6: Proteasome 26S subunit non-ATPase 6.

Expression of PSMD6 protein in normal bile duct epithelium and ICC tissues in the THPA database

This study preliminarily investigated the expression of PSMD6 protein in ICC tissues using the THPA database. We observed that the protein was distributed in the cell membrane/cytoplasm/nucleus of ICC tissues (antibodies HPA036921 and HPA036922), showing moderately to strongly positive expression in ICC tissues (Figure 2). Due to the limited number of cases, however, statistical analysis could not be performed.

Figure 2 Expression of proteasome 26S subunit non-ATPase 6 protein in normal intrahepatic bile duct epithelial tissues and intrahepatic cholangiocarcinoma tissues. A: HPA036921 antibody; patient ID: 2429; normal bile duct cells; staining: Negative; intensity: Negative; quantity: 0; B: HPA036922 antibody; patient ID: 3222; normal bile duct cells; staining: Negative; intensity: Negative; quantity: 0. Images represent the tissue at 20 μm magnification; C: HPA036921 antibody; patient ID: 3625; intrahepatic cholangiocarcinoma (ICC) cells; staining: High; intensity: Strong; quantity: > 75%; localization: Cell membrane/cytoplasm; D: HPA036922 antibody; patient ID: 3625; ICC cells; staining: Moderate; intensity: Moderate; quantity: > 75%; localization: Nucleus. Images represent the tissue at 20 μm magnification.

Expression of PSMD6 protein in normal bile duct epithelium and ICC tissues

This study validated the expression of the PSMD6 protein in ICC and adjacent non-cancerous tissues. PSMD6 protein staining in adjacent non-cancerous tissues showed negative or weakly positive staining (Figure 3A), whereas it exhibited moderately to strongly positive staining in ICC tissues (Figure 3B). To quantitatively analyse the expression of PSMD6 in ICC tissues, a violin plot and ROC curve were generated. The results indicate significantly higher expression of the PSMD6 protein in ICC tissues than in adjacent non-cancerous tissues (P < 0.001) (Figure 4A). ROC curve analysis confirmed the significant overexpression of PSMD6 in ICC tissues, with an area under the curve of 0.999 (95% confidence interval: 0.998-1.000) (Figure 4B). To elucidate the clinicopathological relevance of PSMD6 protein in ICC patients, this study evaluated the relationship between PSMD6 protein expression and clinicopathological characteristics through univariate analysis. The results showed no significant association between PSMD6 protein expression and patient age, gender, pathological grade, or TNM stage (P > 0.05).

Figure 3 Expression of proteasome 26S subunit non-ATPase 6 protein in adjacent non-tumor tissue of intrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma tissue. A: Images are derived from adjacent non-tumor tissue samples of intrahepatic cholangiocarcinoma, representing the tissue at 50 μm magnification; B: Images are derived from intrahepatic cholangiocarcinoma samples, representing the tissue at 50 μm magnification.

Figure 4 High expression of proteasome 26S subunit non-ATPase 6 protein in intrahepatic cholangiocarcinoma. A: Violin plot; B: Receiver operating characteristic curve. PSMD6: Proteasome 26S subunit non-ATPase 6; FPR: False positive rate.

DISCUSSION

Currently, no studies in the literature report the expression and clinicopathological significance of the PSMD6 protein in ICC. The present research for the first time used the CRISPR knockout screening technology to clarify the impact of the PSMD6 gene on the growth of ICC cells at the cellular level. At the protein level, we also evaluated PSMD6 protein expression in ICC tissues through IHC and explored its association with ICC patients’ clinical pathological characteristics, providing a potential molecular indicator for the diagnosis, treatment, and prognosis assessment of ICC patients.

Previous studies have indicated that PSMD6 is closely associated with the occurrence and progression of several tumours, potentially playing a significant role in these processes. Akbari et al[18] used real-time polymerase chain reaction to assess the gene expression profile in granulosa cells in 33 patients with polycystic ovary syndrome, revealing a significant decrease in PSMD6 expression, which suggests that gene expression changes may have important implications for patient fertility. Zhou et al[19] conducted survival analysis and established a correlation between PSMD6 expression levels and postoperative prognosis in patients with pancreatic ductal adenocarcinoma, using co-expression and interaction network analysis to explore the potential biological role of PSMD6 in postoperative pancreatic ductal adenocarcinoma patients. The findings suggest that PSMD6 may serve as a potential prognostic and diagnostic biomarker for early-stage postoperative pancreatic ductal adenocarcinoma patients and may play a crucial role in disease development through regulating key biological processes, such as tumor protein p53 (TP53), cyclin dependent kinase inhibitor 2A, MYC proto-oncogene, BHLH transcription factor (MYC), DNA repair, KRAS proto-oncogene, GTPase (KRAS), cell cycle checkpoints, nuclear factor kappa B (NF-κB) inducing kinase, the NF-κB signaling pathway, and proteasomes. Zhang et al[20] revealed through gene enrichment analysis that PSMD6 may activate the wingless-related integration site signaling pathway by degrading AXIN protein, providing a new perspective for understanding the molecular mechanisms of PSMD6 in lung adenocarcinoma development.

The CRISPR knockout screening method has become an essential tool in gene function research, combining the CRISPR/Cas9 gene editing technology with high-throughput screening to study the function of specific genes in cells or organisms[21-23]. However, changes at the gene level are not always directly reflected at the protein level[24,25], thus requiring further validation to ensure reliability and accuracy. The validation of protein expression through IHC provides a more comprehensive understanding of the role and biological function of genes in tumors[26]. Furthermore, IHC enables a visual demonstration of the specific expression location and distribution of proteins in tissues, which is crucial for a deeper understanding of their role in tumour progression[27]. This study’s results from IHC, violin plot, and ROC curve analysis all suggest high expression of PSMD6 protein in ICC tissues, providing compelling justification for further exploration of its biological functions and potential mechanisms in ICC.

CONCLUSION

The elevated expression of PSMD6 in ICC suggests its potential role as a key protein promoting the onset and progression of ICC. However, this study was limited by the lack of extensive population data validation, and the association between its identified expression levels and patients’ clinicopathological features requires further investigation. Moreover, the specific biological mechanisms of PSMD6 in ICC have not been thoroughly explored. Therefore, future studies should expand the sample size to delve deeper into the biological functions of PSMD6 in ICC and its potential value as a therapeutic target.

ACKNOWLEDGEMENTS

We would like to thank “Guangxi Zhuang Autonomous Region Clinical Medicine Research Center for Molecular Pathology and Intelligent Pathology Precision Diagnosis” for providing technical support.