RPS21 Enhances hepatocellular carcinoma development through GPX4 stabilization

Highlights

• •Upregulation of RPS21 promotes the migration and metastasis and inhibit ferroptosis of Hepatocellular Carcinoma (HCC) in vitro and in vivo.
• •RPS21 can reduce the ubiquitination level of GPX4, thereby stabilizing the expression of GPX4.
• •RPS21 shows promise as a novel therapeutic target for HCC based on its ability to enhance ferroptosis resistance in HCC cells.

Abstract

The study highlights that RPS21, a gene encoding a component of the 40S ribosomal subunit, plays an oncogenic role in hepatocellular carcinoma (HCC) and may influence tumor aggressiveness by affecting antioxidant capacity. RPS21 was found to be upregulated in HCC through RNA-sequencing of clinical samples and analysis of the TCGA database. Kaplan-Meier survival analyses linked higher RPS21 expression to lower survival rates across multiple metrics (OS, PFS, RFS, DSS). Mutation analysis via the cBioPortal showed that primarily amplifications in RPS21 are associated with a poorer prognosis. Tissue microarrays confirmed higher RPS21 levels in tumor samples, which were associated with more advanced clinical stages and grades. Experimental interventions involving lentiviral knockdown or overexpression of RPS21 significantly altered HCC cell proliferation and migration. These findings were supported by mouse models, which showed impacts on tumor growth and metastasis. Further mechanistic studies indicated that RPS21 modulates the ubiquitination and stability of GPX4, a key player in ferroptosis and oxidative stress regulation in HCC cells. This comprehensive study, which merges bioinformatic analysis with laboratory research, positions RPS21 as a viable target for HCC therapy and opens new pathways for understanding and treating liver cancer.

Graphical abstract

Introduction

Hepatocellular carcinoma (HCC) is a major global health concern, ranking as the second leading cause of cancer-related deaths worldwide [1]. It accounts for 85–90 % of primary liver cancer cases, with a particularly high incidence in China [2]. The etiology of HCC is diverse, with hepatitis B virus (HBV) infection being the primary risk factor in China, alongside other contributors such as hepatitis C virus (HCV), alcohol consumption, and non-alcoholic steatohepatitis (NASH) [3]. Despite advances in diagnostic and therapeutic approaches, the prognosis for HCC patients remains poor, largely due to the high incidence of post-surgical metastasis and drug resistance, Current standard treatment for advanced HCC includes sorafenib, a multi-tyrosine kinase inhibitor. However, its efficacy is limited by the development of resistance, highlighting the need for more effective therapeutic strategies [4]. Increasing attention is being paid to the molecular mechanisms underlying HCC. Mahshid Deldar Abad Paskeh and colleagues have indicated that Wnt/β-catenin signaling contributes to drug resistance and immune evasion in HCC, suggesting future research should aim at developing targeted therapies for this pathway [5]. Recently a growing number of studies are emphasizing the critical importance of ferroptosis, a regulated cell death mechanism driven by iron-dependent lipid peroxidation, especially concerning the progression of HCC and its therapeutic responses [6]. Especially the Nrf2/ARE signaling pathway plays a significant role in the progression of HCC and may serve as a crucial therapeutic target in future treatments [7]. Therefore, it is crucial to identify potential oncogenic targets for HCC by investigating the carcinogenic mechanisms, particularly focusing on the ferroptosis mechanisms in HCC cells.

Recent research has focused on exploring the roles of various molecular targets, including ribosomal proteins (RPs) and their homologous analogues, in HCC progression and drug response [8,9]. These studies aim to uncover new therapeutic targets and strategies to overcome the limitations of current treatments. In this study, we investigated the expression and oncogenic mechanisms of RPS21 in hepatocellular carcinoma. RPS21, encoding the 40S ribosomal protein S21, is a crucial component of the small ribosomal subunit and plays a vital role in protein synthesis [10]. Recent studies have highlighted the significance of ribosomal proteins in various human diseases, particularly in cancer development and progression. In the context of cancer research, RPS21 has emerged as a potential oncogene in several malignancies. Notably, it has been identified as a candidate diagnostic and prognostic biomarker for prostate cancer, with upregulation observed in malignant prostate tissues [11]. The oncogenic potential of RPS21 has also been explored in osteosarcoma (OS), where it was found to be overexpressed compared to normal samples. Functional studies in OS cells demonstrated that knockdown of RPS21 significantly inhibited proliferation and migration capabilities, indicating its role in promoting tumor growth and metastasis [12]. Previous research by Török and colleagues has elucidated the functional role of RPS21 as a component involved in the initiation of protein synthesis [10]. Their findings indicate that RPS21 exhibits an association with the native 40S ribosomal subunits, suggesting its participation in the early stages of translation. The Genetic alterations in key translational components, particularly ribosomal proteins, can contribute to carcinogenesis [13,14]. Therefore, this study investigates the oncogenic mechanisms of RPS21 in HCC.

In our study, we identified the potential oncogene RPS21 in HCC through transcriptomic sequencing and validation using bioinformatics databases. Furthermore, we confirmed the promoting effect of RPS21 on the proliferation and migration of HCC cells through gain-loss functional assays, particularly in subcutaneous tumor and lung metastasis mouse models, where we established the oncogenic role of RPS21 in vivo. Subsequent transcriptomic sequencing revealed that RPS21 primarily influences the ferroptosis mechanism in HCC cells. Notably, we found that RPS21 can reduce the ubiquitination levels of GPX4, thereby affecting its expression and influencing the ferroptosis levels in HCC cells. Importantly, GPX4 has the capacity to reverse the oncogenic effects of RPS21.

Results

The expression level of RPS21 is elevated in HCC tissues

We conducted RNA-sequencing on three pairs of HCC tissues and adjacent normal tissues (Fig. 1A), identifying 952 upregulated and 1709 downregulated genes (Fig. 1B). Notably, RPS21 was significantly overexpressed in tumor tissues (Fig. 1B). Further analysis of TCGA-LIHC gene expression data using paired t-tests confirmed RPS21 upregulation in HCC (Fig. 1C). Pan-cancer analysis revealed RPS21 overexpression across multiple human cancers, including CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, and THCA (Fig. 1D). Integrating TCGA-LIHC clinical data, we performed survival analyses and generated Kaplan-Meier curves. Results demonstrated that high RPS21 expression significantly correlated with poor patient outcomes across multiple survival metrics: overall survival (OS, HR=1.44, P = 0.055, Fig. 1E), progression-free survival (PFS, HR=1.62, P = 0.0015, Fig. 1F), recurrence-free survival (RFS, HR=1.72, P = 0.0014, Fig. 1G), and disease-specific survival (DSS, HR=1.71, P = 0.017, Fig. 1H). Univariate and multivariate regression analyses of TCGA-LIHC data revealed that tumor T stage and RPS21 expression serve as independent prognostic indicators for HCC patient outcomes (Table 1). Further investigation using the cBioPortal database revealed that RPS21 amplification was the predominant genetic alteration (Fig. 1I). Importantly, the presence of RPS21 amplification was significantly associated with unfavorable clinical prognosis in HCC patients (Fig. 1J). These findings collectively underscore the potential oncogenic role of RPS21 in HCC and its significance as a prognostic biomarker, warranting further investigation into its molecular mechanisms and therapeutic potential.

Fig. 1.

A. The heatmap illustrates high-throughput sequencing data from three paired HCC samples and their adjacent non-tumor tissues. B. The volcano plot illustrates the number of up- and down-regulated genes identified through high-throughput sequencing of three paired HCC samples. C. Analysis of the TCGA-LIHC dataset using unpaired t-tests revealed significantly elevated RPS21 expression in HCC tissues compared to normal samples. D. Analysis of the TCGA pan-cancer dataset revealed significantly elevated RPS21 expression across multiple cancer types, including HCC, compared to normal tissues. E. Elevated expression of RPS21 is significantly associated with poorer overall survival (OS) in patients with HCC. F. Increased expression of RPS21 is significantly correlated with reduced progression-free survival (PFS) in patients with HCC. G. Patients with hepatocellular carcinoma exhibiting elevated RPS21 expression demonstrate significantly shorter recurrence-free survival (RFS). H. Elevated RPS21 expression is significantly associated with decreased disease-specific survival (DSS) in patients with HCC. I. Analysis using the cBioPortal online database revealed that amplification is the predominant mutation type for RPS21 in cancer samples. J. Analysis using the cBioPortal database revealed that patients with RPS21 mutations exhibit poorer survival outcomes compared to those without mutations.

Table 1.

Univariate and multivariate regression analysis of clinical-pathological features and RPS21 expression on HCC patients’ prognosis using TCGA-LIHC data.

Characteristics	Total(N)	Univariate analysis		Multivariate analysis
		Hazard ratio (95 % CI)	P value	Hazard ratio (95 % CI)	P value
Pathologic T stage	370
T1	183	Reference		Reference
T2	94	1.431 (0.902–2.268)	0.128	1.544 (0.857–2.781)	0.148
T3	80	2.674 (1.761–4.060)	< 0.001	3.245 (1.942–5.421)	< 0.001
T4	13	5.386 (2.690–10.784)	< 0.001	5.918 (2.154–16.262)	< 0.001
Pathologic N stage	258
N0	254	Reference
N1	4	2.029 (0.497–8.281)	0.324
Pathologic M stage	272
M0	268	Reference		Reference
M1	4	4.077 (1.281–12.973)	0.017	1.165 (0.283–4.800)	0.832
Gender	373
Female	121	Reference
Male	252	0.793 (0.557–1.130)	0.200
Age	373
≤ 60	177	Reference
> 60	196	1.205 (0.850–1.708)	0.295
RPS21	373
Low	187	Reference		Reference
High	186	1.416 (1.001–2.001)	0.049	1.758 (1.127–2.740)	0.013

Clinical validation of RPS21 overexpression in HCC

To further validate our findings, we conducted an immunohistochemical (IHC) analysis using a tissue microarray comprising 80 paired HCC specimens (Fig. 2A). The results demonstrated significantly elevated expression of RPS21 in HCC tissues compared to adjacent normal tissues (Fig. 2B-D). Notably, RPS21 expression positively correlated with advancing tumor stage (Fig. 2C-E). We corroborated these findings by analyzing TCGA-LIHC data using the UALCAN online platform, which confirmed the positive association between RPS21 expression and both clinical stage and histological grade in HCC patients (Fig. 2F-G). These consistent results from multiple approaches underscore the potential clinical significance of RPS21 in HCC progression. Meanwhile, Chi-square analysis of TCGA-LIHC data revealed a significant association between RPS21 expression and pathological grade in hepatocellular carcinoma patients (Table 2).

Fig. 2.

A. Immunohistochemical analysis was performed on 80 paired HCC samples, comprising tumor tissues and adjacent non-tumor tissues. B. Statistical analysis of immunohistochemical scores revealed significantly higher RPS21 staining intensity in tumor tissues compared to corresponding adjacent non-tumor tissues. C. Statistical analysis of immunohistochemical scores revealed that RPS21 staining intensity increased significantly with advancing tumor stages in cancerous tissues. D. Representative immunohistochemical images demonstrate differential RPS21 staining intensity between tumor tissues and corresponding adjacent non-tumor tissues. E. Representative immunohistochemical images illustrate differential RPS21 staining intensities across tumor tissues of varying stages. F. Analysis using the UALCAN database confirmed that RPS21 expression progressively increases with advancing tumor grades. G. Analysis using the UALCAN database demonstrated a positive correlation between RPS21 expression levels and advancing tumor stages.

Table 2.

Clinical baseline data table of TCGA-LIHC patient information used in this study.

Characteristics	Low expression of RPS21	High expression of RPS21	P value
N	187	187
Pathologic T Stage, n (%)			0.102
T1	102 (27.5 %)	81 (21.8 %)
T2	43 (11.6 %)	52 (14 %)
T3&T4	41 (11.1 %)	52 (14 %)
Pathologic M Stage, n (%)			0.800
M0	118 (43.4 %)	150 (55.1 %)
M1	1 (0.4 %)	3 (1.1 %)
Pathologic Stage, n (%)			0.341
Stage I	93 (26.6 %)	80 (22.9 %)
Stage II	39 (11.1 %)	48 (13.7 %)
Stage III	37 (10.6 %)	48 (13.7 %)
Stage IV	2 (0.6 %)	3 (0.9 %)
Gender, N (%)			0.439
Female	57 (15.2 %)	64 (17.1 %)
Male	130 (34.8 %)	123 (32.9 %)
Race, N (%)			< 0.001
Asian	53 (14.6 %)	107 (29.6 %)
Black or African American	13 (3.6 %)	4 (1.1 %)
White	116 (32 %)	69 (19.1 %)
Histologic Grade, N (%)			< 0.001
G1	35 (9.5 %)	20 (5.4 %)
G2	106 (28.7 %)	72 (19.5 %)
G3	43 (11.7 %)	81 (22 %)
G4	2 (0.5 %)	10 (2.7 %)

RPS21 significantly suppresses HCC proliferation

Western blot analysis revealed highest RPS21 expression in SKP-Hep-1 cells and lowest in Huh7 cells (Supplementary Figure 1 A). Consequently, these two cell lines were selected to establish stable knockdown and overexpression models, respectively. Subsequent Western blots confirmed successful RPS21 modulation in both cell lines (Supplementary Figure 1B-C). Subsequent cellular experiments revealed significant alterations in proliferative capacity. Clonogenic assays demonstrated that RPS21 knockdown markedly reduced colony formation (Fig. 3A-B), while RPS21 overexpression significantly enhanced it (Fig. 3A-B). The EdU incorporation assays showed a substantial decrease in proliferating cells following RPS21 knockdown, whereas RPS21 overexpression led to a notable increase (Fig. 3C-F). Furthermore, CCK-8 assays corroborated these findings, indicating diminished cell viability upon RPS21 knockdown and enhanced proliferative activity with RPS21 overexpression (Fig. 3G-H).

Fig. 3.

A. Clonogenic assays in SKP-hep-1 and Huh7 HCC cells revealed altered colony formation following RPS21 knockdown and overexpression. B. Statistical analysis of clonogenic assays revealed alterations in colony formation following RPS21 knockdown and overexpression in SKP-hep-1 and Huh7 HCC cells. C. EdU assays in SKP-hep-1 HCC cells demonstrated alterations in proliferating cell numbers following RPS21 knockdown and overexpression. D. Statistical analysis of EdU assays in SKP-Hep-1 HCC cells revealed changes in proliferating cell numbers following RPS21 knockdown and overexpression. E. EdU assays in Huh7 HCC cells demonstrated alterations in proliferating cell populations following RPS21 knockdown and overexpression. F. Statistical analysis of EdU assays in Huh7 HCC cells revealed changes in proliferating cell numbers following RPS21 knockdown and overexpression. G. CCK-8 assays were conducted to assess changes in SKP-hep-1 HCC cell viability following RPS21 knockdown and overexpression. H. Cell viability changes in Huh7 HCC cells following RPS21 knockdown and overexpression were assessed using CCK-8 assays.

RPS21 significantly suppresses HCC migration

We successfully established stable RPS21 knockdown and overexpression cell lines in SK-Hep-1 and Huh7 HCC cells using lentiviral constructs. Subsequent cellular experiments revealed significant alterations in metastatic potential. Transwell assays demonstrated that RPS21 knockdown markedly reduced both migration and invasion of HCC cells (Fig. 4A-D), while RPS21 overexpression significantly enhanced these processes (Fig. 4A-D). Wound healing assays corroborated these findings, showing a substantial decrease in cell migration capacity following RPS21 knockdown, whereas RPS21 overexpression led to a notable increase in migratory ability (Fig. 4E-H). Furthermore, we collected tumor tissues and corresponding adjacent non-tumor tissues from HCC patients. Immunofluorescence analysis of these samples revealed a significant negative correlation between RPS21 expression and E-cadherin levels. Conversely, RPS21 expression showed strong positive correlations with both N-cadherin and Vimentin expression (Fig. 4I).

Fig. 4.

A. Transwell assays evaluated changes in SKP-hep-1 HCC cell proliferation and migration following RPS21 knockdown and overexpression. B. Statistical analysis bar graph depicting changes in SKP-hep-1 HCC cell proliferation and migration following RPS21 knockdown and overexpression, as assessed by Transwell assays. C. Transwell assays evaluated alterations in Huh7 HCC cell proliferation and migration following RPS21 knockdown and overexpression. D. Bar graph depicting statistical analysis of changes in Huh7 HCC cell proliferation and migration following RPS21 knockdown and overexpression, as assessed by Transwell assays. E. Bar graph illustrating statistical analysis of SKP-hep-1 HCC cell migration changes following RPS21 knockdown and overexpression, as assessed by wound healing assays. F. Wound healing assays evaluated changes in SKP-hep-1 HCC cell migration following RPS21 knockdown and overexpression. G. Statistical analysis of wound healing assays revealed changes in Huh7 HCC cell migration following RPS21 knockdown and overexpression. H. Wound healing assays evaluated alterations in Huh7 HCC cell migration following RPS21 knockdown and overexpression. I. Immunofluorescence analysis of E-cadherin, N-cadherin, and Vimentin expression in adjacent non-tumor tissues and HCC tissues with high and low RPS21 expression.

In vivo studies confirm RPS21 promotes tumor growth and metastasis

To further validate our in vitro findings, we established subcutaneous xenograft and lung metastasis models in mice. In the subcutaneous xenograft model, we observed that RPS21 knockdown significantly impeded tumor growth kinetics, resulting in markedly reduced final tumor weights upon harvest (Fig. 5A–C). Conversely, RPS21 overexpression substantially accelerated tumor growth, leading to significantly increased final tumor weights (Fig. 5A–C). Complementing these findings, our tail vein injection lung metastasis model yielded concordant results. RPS21 knockdown led to a notable reduction in the number of metastatic lung nodules, while RPS21 overexpression resulted in a significant increase in metastatic burden (Fig. 5D–G). These in vivo observations strongly corroborate our in vitro data, collectively suggesting that RPS21 plays a crucial role in both primary tumor growth and metastatic potential of hepatocellular carcinoma. The consistent effects observed across multiple experimental models underscore the robustness of RPS21′s influence on tumor progression. Our findings not only validate the oncogenic properties of RPS21 in a physiologically relevant context but also highlight its potential as a therapeutic target. The ability of RPS21 modulation to affect both local tumor growth and distant metastasis formation suggests that targeting this protein could potentially address multiple aspects of cancer progression simultaneously.

Fig. 5.

A. Images of harvested tumors from the established subcutaneous xenograft mouse model. B. Statistical analysis of differential subcutaneous tumor growth rates among groups in the mouse xenograft model. C. Statistical analysis of final tumor weight differences among groups in the subcutaneous xenograft mouse model. D. Statistical analysis of differential lung metastatic nodule formation among groups in the mouse tail vein injection lung metastasis model. E. Representative macroscopic images of lung metastatic nodules from different groups in the mouse tail vein injection lung metastasis model. F. In vivo small animal imaging was used to detect lung metastases across different groups in the mouse tail vein injection lung metastasis model. G. Hematoxylin and eosin staining of lung tissues revealed differences in metastatic nodule numbers among groups in the mouse tail vein lung metastasis model.

RPS21 inhibits HCC ferroptosis by reducing GPX4 ubiquitination

RNA sequencing analysis of stable RPS21 knockdown cell lines, SKP-Hep-1 and Huh7, revealed significant transcriptional changes. In SKP-Hep-1 cells, RPS21 knockdown resulted in the upregulation of 489 genes and downregulation of 361 genes (Fig. 6A-B). Similarly, in Huh7 cells, 264 genes were upregulated and 193 genes were downregulated following RPS21 knockdown (Fig. 6D–E). Notably, gene set enrichment analysis indicated that differentially expressed genes in both cell lines were predominantly enriched in ferroptosis-related pathways, suggesting a potential role for RPS21 in modulating ferroptosis in HCC cells (Fig. 6C and F). To further investigate the relationship between RPS21 and ferroptosis, we examined the expression of GPX4, a key regulator of ferroptosis. Western blot analysis demonstrated that RPS21 knockdown led to a decrease in GPX4 expression in both SKP-Hep-1 and Huh7 cells, while RPS21 overexpression resulted in increased GPX4 levels. These findings indicate a positive correlation between RPS21 and GPX4 expression (Fig. 6G–H). Co-immunoprecipitation experiments revealed an inverse relationship between RPS21 expression and GPX4 ubiquitination levels. Higher RPS21 expression was associated with reduced GPX4 ubiquitination, whereas RPS21 knockdown resulted in increased GPX4 ubiquitination (Fig. 6I–J). These results suggest that RPS21 may regulate ferroptosis in HCC cells by modulating GPX4 protein stability. To assess the functional consequences of RPS21-mediated regulation of GPX4, we examined cellular reactive oxygen species (ROS) levels using flow cytometry. RPS21 knockdown significantly increased intracellular ROS levels, a hallmark of ferroptosis. Importantly, GPX4 overexpression was able to reverse the elevated ROS levels induced by RPS21 knockdown, further supporting the functional link between RPS21 and GPX4 in regulating ferroptosis (Fig. 6K–L). Additionally, immunofluorescence microscopy using the TMRE mitochondrial membrane potential indicator revealed that RPS21 knockdown led to a significant decrease in mitochondrial membrane potential, another characteristic feature of ferroptosis. Notably, GPX4 overexpression was able to rescue this reduction in mitochondrial membrane potential (Fig. 6M–N), providing further evidence for the role of the RPS21-GPX4 axis in regulating ferroptosis in HCC cells.

Fig. 6.

A. A heatmap illustrating the differential gene expression profiles between the control group and RPS21 knockdown group in SKP-HEP-1 cells. B. A volcano plot illustrating the number of upregulated and downregulated differentially expressed genes in SKP-HEP-1 cells, comparing the control group to the RPS21 knockdown group based on sequencing data. C. KEGG pathway enrichment analysis was performed on differentially expressed genes identified through sequencing of SKP-HEP-1 cells in the control and RPS21 knockdown groups, revealing downstream signaling pathways. D. A heatmap illustrating the distribution of differentially expressed genes between the control group and RPS21 knockdown group in Huh7 cells. E. A volcano plot illustrating the number of upregulated and downregulated differentially expressed genes identified through sequencing of Huh7 cells in the control and RPS21 knockdown groups. F. KEGG pathway enrichment analysis was performed on differentially expressed genes identified through sequencing of Huh7 cells in the control and RPS21 knockdown groups, elucidating downstream signaling pathways. G. Western blot analysis revealed that knockdown of RPS21 in SKP-HEP-1 and Huh7 cells resulted in a concomitant decrease in GPX4 expression. H. Western blot analysis revealed that overexpression of RPS21 in SKP-HEP-1 and Huh7 cells led to a concomitant increase in GPX4 expression. I. Co-immunoprecipitation analysis revealed that overexpression of RPS21 using varying plasmid concentrations in SKP-HEP-1 and Huh7 cells resulted in a dose-dependent decrease in GPX4 ubiquitination levels. J. Co-immunoprecipitation analysis revealed that lentiviral-mediated knockdown of RPS21 in SKP-HEP-1 and Huh7 cells resulted in a concomitant increase in GPX4 ubiquitination levels. K. Flow cytometry was employed to measure reactive oxygen species (ROS) levels of SKP-HEP-1 cell in three experimental groups: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4).L. Flow cytometry was employed to measure reactive oxygen species (ROS) levels of SKP-HEP-1 cell in three experimental groups: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4). M. Cellular immunofluorescence analysis revealed changes in mitochondrial membrane potential (TMRE) across three experimental groups in SKP-HEP-1 cells: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4). N. Cellular immunofluorescence analysis was employed to visualize changes in mitochondrial membrane potential (TMRE) in Huh7 cells across three experimental groups: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4).

GPX4 reverses RPS21-induced alterations in cellular proliferation and migration capacity

To elucidate the role of GPX4 in modulating RPS21-mediated oncogenic activities in HCC, we conducted a comprehensive series of experiments. Our investigation focused on assessing the impact of GPX4 expression modulation on RPS21-induced oncogenic properties. Transwell assays were employed to evaluate the influence of GPX4 on RPS21-induced migration and invasion in HCC cells (Fig. 7A–D). The results, illustrated in, demonstrated that GPX4 significantly attenuated the pro-migratory and pro-invasive effects of RPS21. Corroborating these findings, wound healing assays yielded consistent outcomes, confirming GPX4′s capacity to counteract RPS21-mediated enhancement of cell motility (Fig. 7E–H). To assess the impact on cellular proliferation, we performed colony formation assays on the modified cell lines. As shown in, GPX4 expression effectively mitigated the RPS21-induced proliferative advantage in HCC cells. Collectively, these findings suggest that GPX4 plays a crucial role in counteracting the oncogenic properties conferred by RPS21 in HCC (Fig. 7I–L). GPX4 appears to function as a modulator of RPS21-mediated effects on cellular migration, invasion, and proliferation, potentially offering a novel therapeutic avenue for HCC treatment. The Graphical Abstract of our study is presented in Fig. 8.

Fig. 7.

A. Transwell assays were conducted to evaluate changes in cell migration and invasion capabilities across three experimental groups in SKP-HEP-1 cells: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4). B. Statistical analysis of cell migration and invasion was performed and presented as a bar graph, comparing three experimental groups in SKP-HEP-1 cells: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4). C. Transwell assays were conducted to evaluate changes in cell migration and invasion capabilities across three experimental groups in Huh7 cells: the control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4). D. Statistical analysis of cell migration and invasion was conducted and presented as a bar graph, comparing three experimental groups in Huh7 cells: the control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4). E. Scratch wound assays were performed to evaluate changes in cell migration across three experimental groups in SKP-HEP-1 cells: the control group (shCon+pc-Con), the RPS21 knockdown group (shRPS21+pc-Con), and the RPS21 knockdown with GPX4 overexpression group (shRPS21+pc-GPX4). F. Statistical analysis of cell migration in SKP-HEP-1 cells across control, RPS21 knockdown, and RPS21 knockdown/GPX4 overexpression groups, presented as bar graphs. G. Scratch wound assays demonstrated changes in cell migration among three Huh7 cell groups: control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4). H. Statistical analysis of cell migration in Huh7 cells was performed and presented as bar graphs, comparing three experimental groups: control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4). I. Colony formation assays revealed changes in clonogenic potential among three SKP-HEP-1 cell groups: control (shCon+pc-Con), RPS21 knockdown (shRPS21+pc-Con), and RPS21 knockdown with GPX4 overexpression (shRPS21+pc-GPX4). J. Statistical analysis of colony formation in SKP-HEP-1 cells was conducted and presented as bar graphs, comparing three experimental groups: control (shCon+pc-Con), RPS21 knockdown (shRPS21+pc-Con), and RPS21 knockdown with GPX4 overexpression (shRPS21+pc-GPX4). K. Colony formation assays revealed changes in clonogenic potential among three Huh7 cell groups: control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4). L. Statistical analysis of colony formation in Huh7 cells was conducted and presented as bar graphs, comparing three experimental groups: control (OV-con+si-con), RPS21 overexpression (OV-RPS21+si-con), and RPS21 overexpression with GPX4 knockdown (OV-RPS21+si-GPX4).

Fig. 8.

The Graphical Abstract of the study.

Discussion

Our study reveals a novel mechanism by which RPS21 promotes HCC progression through the inhibition of ferroptosis. We demonstrate that RPS21 reduces the ubiquitination levels of GPX4, thereby stabilizing this key regulator of ferroptosis and consequently enhancing HCC cell proliferation and migration. The role of ribosomal proteins in cancer development and progression has gained increasing attention in recent years [15]. While traditionally viewed as components of the protein synthesis machinery, ribosomal proteins are now recognized to have extra-ribosomal functions that can significantly impact cellular processes [16]. Recent literature has highlighted the crucial role of ribosomal proteins (RPs) in tumor metastasis and drug resistance [17]. Gaining a deeper understanding of the mechanisms that contribute to resistance against therapies targeting ribosome biogenesis offers valuable insights into the molecular mechanisms of cancer vulnerability associated with Ribosomopathies. This knowledge also presents significant clinical implications for the treatment of cancer [15]. Notably, a 2020 study by Richard Y Ebright et al. revealed that the expression and translation of RPs significantly promote breast cancer metastasis [18]. RPs are often overactivated in cancer cells to fulfill the increased demands of protein synthesis necessary for self-biosynthesis and metabolic processes. This dysregulation is particularly pronounced in various stages of tumorigenesis and can significantly affect both the speed and quality of protein synthesis. Consequently, such alterations can ultimately impact the transcriptome associated with the generation of relevant proteomes [19]. Recent studies have highlighted that RPS15, a member of the RPs family, interacts with IGF2BP1, thereby promoting the progression of esophageal squamous cell carcinoma (ESCC) through the p38 MAPK signaling pathway. Furthermore, RPS15 inhibitors may represent promising candidates for the development of novel anti-ESCC therapeutics [20]. Recent research in the field of non-small cell lung cancer (NSCLC) conducted by Jie Chen et al. has identified RPL11, a member of the ribosomal protein (RP) family, as a key regulator of endoplasmic reticulum stress (ERS) and autophagy in lung cancer cells. This regulatory role promotes the progression of NSCLC by enhancing the proliferation of lung cancer cells [21]. Xie and colleagues have also reviewed the exploration of ribosomal proteins in the disease mechanisms of HCC, suggesting that ribosomal proteins play important roles in the development and progression of HCC [22]. Recent studies have revealed that ribosomal protein S7 (RPS7) is significantly elevated in HCC tissues and is strongly associated with poor survival outcomes in HCC patients. Furthermore, RPS7 has been shown to enhance HCC metastasis through the LOXL2/ITGB1 axis, providing new insights for the development of molecular therapeutics targeting HCC [23]. Li et al. suggested that RPS24 may serve as a potential negative prognostic biomarker for HCC, as it promotes cell proliferation and contributes to the establishment of an immunosuppressive microenvironment. Targeting RPS24 could represent a promising therapeutic strategy for the management of HCC [24]. Zhou et al. also recognized the therapeutic potential of targeting RPs and their regulatory networks to develop more effective treatment approaches, with particular emphasis on the intricate involvement of RPs like RPS5 in the malignant development of HCC [25]. Nevertheless, the specific functions and mechanisms of RPS21, a member of the ribosomal protein family, in the context of HCC remain unreported in the existing literature. This gap in the literature underscores the need for further investigation into the potential functions of RPS21 in HCC pathogenesis. Our findings add to this growing body of evidence by elucidating a novel function of RPS21 in HCC.

RPS21 has been previously implicated in other cancer types, such as osteosarcoma, where its down-regulation was found to inhibit invasive behavior through the inactivation of the MAPK pathway [12]. Furthermore, studies have demonstrated that RPL21L and RPS21 exhibit elevated expression in prostate cancer (PCa), with notably higher levels observed in cases of high Gleason grade compared to those of low Gleason grade [11]. Experimental investigations using in vitro models have elucidated that these ribosomal proteins (RPs) enhance cellular proliferation and metastatic potential while simultaneously suppressing apoptotic processes in PCa cell lines. These findings are corroborated by Fan et al., who identified numerous crucial genes and signaling cascades implicated in PCa pathogenesis [26]. The consistent overexpression of these RPs across multiple studies underscores their potential significance in PCa progression and their promise as molecular indicators for disease prognosis. Our study extends these findings to HCC and reveals a different mechanism of action, centered on the regulation of ferroptosis. Meanwhile, it is noteworthy that there have been no reports to date examining the relationship between members of the ribosomal protein family, particularly RPS21, and ferroptosis in tumor cells. Our study represents the first investigation addressing this gap, providing significant insights into the role of RPS21 in ferroptosis in HCC cells and elucidating its underlying mechanisms.

Ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, has emerged as a critical process in cancer biology [27]. Our results highlight the importance of ferroptosis resistance in HCC progression, aligning with previous studies that have shown the significance of this pathway in HCC [28]. By demonstrating that RPS21 can modulate ferroptosis sensitivity through GPX4 stabilization, we provide new insights into the complex regulatory networks governing this process in HCC. GPX4, as a key regulator of ferroptosis, plays a central role in protecting cells from lipid peroxidation-induced death [29]. Especially in HCC, it plays an increasingly important role. Recently, Leyi Yao et al. proposed that Plumbagin can degrade GPX4 protein, thereby mediating apoptosis in HCC cells [30]. Xiangying Yan et al. proposed that Pien-Tze-Huang can promote ferroptosis in HCC cells by inhibiting the SLC7A11-GSH-GPX4 axis, thereby exerting potential therapeutic effects in HCC [31]. In a recent study, Feifan Yao et al. utilized CRISPR/Cas9 screening to discover that TRIM34 mediates resistance to ferroptosis in HCC cells. Interestingly, the inhibition of TRIM34 expression was found to enhance the sensitivity of HCC cells to PD-L1 therapy [32]. Jiarui Li et al. discovered that PLAG1 is essential for maintaining redox equilibrium by enhancing the expression of GPX4, an enzyme that plays a key role in reducing lipid peroxides (LPOs). This mechanism ultimately contributes to the inhibition of ferroptosis in HCC [33]. Notably, recent research by Ruogu Pan et al. has established that GPX4 is upregulated in HCC tissues and that it can inhibit ferroptosis in HCC cells through the GRHL3/PTEN/PI3K/AKT axis, thereby promoting HCC metastasis [34]. Our finding that RPS21 enhances GPX4 stability by reducing its ubiquitination levels reveals a previously unknown mechanism of GPX4 regulation. This interaction between RPS21 and GPX4 represents a novel link between ribosomal proteins and ferroptosis regulation, expanding our understanding of both fields. The observed increase in HCC proliferation and migration upon RPS21 overexpression can be attributed to the suppression of ferroptosis. By maintaining higher levels of GPX4, RPS21 enables HCC cells to evade this form of cell death, promoting their survival and aggressive behavior. This finding underscores the importance of ferroptosis resistance as a hallmark of cancer progression and metastasis. Building on previous research, it has been further confirmed that RPS21 holds significant potential as a therapeutic target for HCC. This is achieved by influencing the occurrence of ferroptosis in HCC cells, which subsequently affects their metastasis and invasion. Our results suggest that targeting the RPS21-GPX4 axis could be a promising therapeutic strategy for HCC. Inhibiting RPS21 or disrupting its interaction with GPX4 may sensitize HCC cells to ferroptosis, potentially leading to reduced tumor growth and metastasis. This approach could be particularly valuable in combination with existing therapies, as ferroptosis induction has shown synergistic effects with other treatment modalities in various cancer types. Our study highlights the potential of combining ferroptosis inducers with RPS21 inhibitors as a novel approach for HCC treatment. Such combination therapies could potentially overcome the limitations of current treatments and address the issue of drug resistance, which remains a significant challenge in HCC management.

It is important to note that while our study provides compelling evidence for the role of RPS21 in HCC progression, there are limitations that should be addressed in future research. First, our findings are primarily based on in lung metastasis model and xenograft model. Further validation in more complex in vivo models. Additionally, the exact molecular mechanisms by which RPS21 reduces GPX4 ubiquitination remain to be fully elucidated. Investigating the potential involvement of deubiquitinating enzymes or other regulatory proteins could provide a more comprehensive understanding of this process.

In conclusion, our research unveils a previously unknown function of RPS21 in HCC progression, linking ribosomal proteins to ferroptosis regulation through the stabilization of GPX4. These findings not only expand our understanding of HCC pathogenesis but also open new avenues for therapeutic interventions targeting the RPS21-GPX4 axis in HCC. As we continue to unravel the complex interplay between ribosomal proteins, ferroptosis, and cancer progression, we move closer to developing more effective and targeted treatments for HCC and potentially other malignancies.

Methods

Bioinformatic analysis

To investigate the clinical significance of RPS21 in hepatocellular carcinoma (HCC), a comprehensive bioinformatic analysis was used combing multiple publicly available databases. Genetic mRNA data and corresponding clinical information from HCC patients were extracted from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). Differential expression analysis of RPS21 was performed using the "limma" package in R software (version 4.2.2). To elucidate the prognostic value of RPS21 expression, the Kaplan-Meier plotter (K-M plotter) online tool was used to assess the correlation between RPS21 expression levels and patient survival outcomes. The cBioPortal (http://cbioportal.org) serves as a freely accessible platform for researchers to interactively investigate complex cancer genomics datasets. This comprehensive resource currently offers users the ability to explore and analyze molecular data derived from over 5000 tumor specimens, encompassing 20 distinct cancer studies [35]. By providing an interface for the dynamic exploration of multifaceted genomic information, the cBioPortal database was employed to examine RPS21 gene mutation profiles and their potential impact on HCC patient prognosis. The UALCAN platform serves as a comprehensive resource for cancer researchers, facilitating the analysis and dissemination of transcriptomic, proteomic, and survival data. Leveraging information from The Cancer Genome Atlas (TCGA), this tool enables investigators to examine protein-coding gene expression patterns and their correlation with patient outcomes across 33 distinct cancer types [36]. the relationship between RPS21 expression and various clinical characteristics of HCC patients was explored by using the UALCAN web-based platform. This comprehensive approach allowed us to gain insights into the potential role of RPS21 in HCC pathogenesis and its clinical implications. By integrating data from these diverse bioinformatic resources, the study aimed to provide a thorough and multifaceted analysis of RPS21′s significance in HCC, encompassing its expression patterns, prognostic value, mutation landscape, and associations with clinicopathological features

Clinical samples collecting

The HCC specimens and adjacent non-neoplastic tissues were procured from patients undergoing surgical resection at the Department of Hepato-biliary-pancreatic Surgery, Institute of Hepatobiliary and Pancreatic Diseases. All tissue acquisitions were conducted in strict adherence to ethical guidelines, with written informed consent obtained from each participant.

Post-resection, tissue samples were processed using two distinct protocols to facilitate comprehensive molecular and histological analyses. For transcriptomic profiling, specimens were immediately cryopreserved in liquid nitrogen or stored at −80 °C to preserve RNA integrity. Concurrently, tissue samples were fixed in 4 % paraformaldehyde and embedded in paraffin for subsequent immunohistochemical studies and tissue microarray (TMA) construction. Total RNA was extracted from matched HCC and non-neoplastic tissue pairs from three patients and submitted to Riobio company for high-throughput sequencing analysis. This approach enabled the identification of differentially expressed genes (DEGs) between cancerous and non-cancerous tissues, providing insights into the molecular landscape of HCC. The parallel preparation of formalin-fixed, paraffin-embedded (FFPE) samples facilitated the construction of tissue microarrays, laying the groundwork for future immunohistochemical investigations. This dual-pronged approach allows for the integration of transcriptomic data with protein-level analyses, potentially unveiling novel biomarkers and therapeutic targets in HCC.

Cell culture and shRNA transfection

The human HCC cell lines SKP-hep-1 and Huh7 were procured from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. These cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin solution. All cell cultures were incubated at 37 °C in a humidified atmosphere containing 5 % CO2, adhering to standard cultivation protocols. For genetic manipulation experiments, lentiviral vectors encoding short hairpin RNA (shRNA) targeting RPS21, along with appropriate control vectors, were acquired from Shanghai Genechem Co. Ltd. The SKP-hep-1 and Huh7 cells were subsequently transduced with these lentiviral constructs. Detailed information regarding the RPS21 and GPX4 sequence and shRNA sequences can be found in Supplementary Tables 1, respectively. Following transduction, a selection process was implemented to isolate successfully transfected cells. This was achieved by culturing the cells in medium supplemented with puromycin for a period of 5 days. This rigorous selection procedure ensured the establishment of stable cell lines expressing the desired genetic modifications, thereby facilitating further experimental investigations into the role of RPS21 in HCC pathogenesis.

Immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB)

Western blotting, immunohistochemistry, and immunofluorescence were performed following established protocols as described in the literature [37]. These techniques were implemented adhering to standardized procedures to ensure reproducibility. A comprehensive list of antibodies employed in this investigation is provided in Supplementary Table 2.

Cell proliferation assay

Cell proliferation assays were conducted following established protocols as described in the literature, ensuring reproducibility and adherence to standardized procedures [37].

Cellular migration and invasion assessment

Cell migration and invasion assays were conducted following established protocols, ensuring reproducibility and adherence to standardized procedures [37].

Flow cytometry

Quantification of total reactive oxygen species (ROS) was performed using a commercially available ROS Assay Kit. Following the manufacturer's protocol, the human HCC cell lines SKP-hep-1 and Huh7 were incubated with DCFHDA probe, diluted 1:2000 in serum-free medium, for 15 min at 37 °C, with intermittent mixing every 5 min. Subsequently, the cell suspension underwent centrifugation and three washing cycles with serum-free DMEM medium. The resulting cell pellet was then resuspended in 500 μl of PBS for further analysis. This method enables accurate assessment of intracellular ROS levels in experimental samples.

Co-immunoprecipitation (Co-IP)

Co-immunoprecipitation analyses followed established protocols [37].

In vivo tumor growth and lung metastasis evaluation

Four- to five-week-old nude mice were procured from Cavens Biogle (Changzhou, China) and maintained in a specific pathogen-free facility. ShRNA-transfected HCC cells (SKP-hep-1 and Huh7) were subcutaneously inoculated into the axillary region at a concentration of 4 × 10^6 cells per mouse. Tumor dimensions were measured bi-weekly for 21 days post-injection. Tumor volume was calculated using the formula V = 0.5 × (minor diameter)^² × (major diameter). This experimental design aligns with established protocols for in vivo tumor growth assessment in xenograft models, ensuring reproducibility and adherence to standardized procedures.

Statistical analysis for data interpretation

Statistical analyses were conducted using GraphPad Prism 8 and R (version 4.0.0). All experiments were performed in triplicate, and quantitative data were analyzed using t-tests to evaluate the prognostic significance of RPS21. Results are presented as mean ± standard deviation. Statistical significance was defined as p < 0.05. This approach aligns with established protocols for biostatistical analysis in biomedical research, ensuring reproducibility and adherence to standardized procedures.

Abbreviations

HCC Hepatocellular carcinoma

HBV Hepatitis B virus

HCV Hepatitis C virus

NASH Non-alcoholic steatohepatitis

RPS21 ribosomal protein S21

ROS reactive oxygen species

PCa prostate cancer

RPs ribosomal proteins

TCGA The Cancer Genome Atlas

K-M Kaplan-Meier plotter

IHC Immunohistochemistry

IF Immunofluorescence

WB Western Blotting

Co-IP Co-immunoprecipitation

Experiments with human subjects statement

All the authors ensure that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The manuscript has been in line with the Recommendations for the Conduct, Reporting, Editing and Publication of Scholarly Work in Medical Journals. The representative human populations has been included in the manuscript.

Animal ethics statement

All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). All the authors clearly indicate that the above guidelines have been followed. Animal experiments were conducted in accordance with the guidelines and policies set forth by the Ethics Committee of Jiangsu Kerbio Medical Technology Group Co. Ltd. In this study, a total of 50 nude mice were used, all of which were male in order to exclude the influence of estrogen itself on hepatobiliary tumors.

Declarations

Ethics approval and consent to participate

The acquisition of human tumor specimens for this study received approval from the ethics committee of Changzhou Second People's Hospital, with informed consent obtained from all patients and Nanjing Medical University. Animal experiments were conducted in accordance with the guidelines and policies set forth by the Ethics Committee of Jiangsu Kerbio Medical Technology Group Co. Ltd.

Consent for publication

The publication has obtained consent from all authors.

Funding

The project is funded by Nanjing Medical University Scientific and Technological Development Fund (NMUB20230044).

Data availability statement

The datasets utilized in this study can be made available upon reasonable request from the corresponding author.

CRediT authorship contribution statement

Siyuan Wu: Software, Methodology, Conceptualization. Gaochao Wang: Data curation. Likai Gu: Writing – original draft. Yinjie Zhang: Software, Methodology. Zhihuai Wang: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.