RPLP0 drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy

Introduction

Non-Hodgkin lymphoma (NHL) is a heterogeneous malignancy of the lymphatic system, and dozens of subtypes have been identified to date (1). With the aging of the global population, the incidence burden of NHL is expected to increase substantially, with the number of newly diagnosed cases projected to rise from ~545,000 cases in 2020 to 778,000 cases by 2040(2). Among patients with NHL, 30-40% are of the most prevalent subtype, diffuse large B-cell lymphoma (DLBCL) (3). The incidence of DLBCL varies geographically and increases substantially with age, ranging from ~2.3 to 13.8 cases per 100,000 person-years (4). Elderly individuals, particularly those aged >60 years, are more frequently affected (5). Gene damage, epigenetic instability and long-term immunological activation all contribute to its etiology (6). Together, these elements alter interactions within the tumor microenvironment, B-cell differentiation and survival signal transduction. A notable portion of patients continue to develop primary resistance or relapse despite marked advancements in molecular profiling (7). Rituximab with cyclophosphamide, doxorubicin, vinblastine and prednisone is the standard first-line treatment (8). For high-risk disease subgroups, such as patients with aggressive biological characteristics or a poor response to immunochemotherapy, the results are still disappointing (9).

A notable factor influencing stress adaptation in cancer is autophagy, a conserved lysosome-dependent catabolic process (10). Autophagy promotes metabolic flexibility, limits oxidative stress and modifies immune-related signals by removing damaged organelles and protein aggregates (11). Impaired autophagy has been associated with worse outcomes following immunochemotherapy in DLBCL. According to Xu et al (12) the paired box 5-driven long non-coding RNA (lncRNA) ARRDC1-AS1 enhances autophagy by regulating autophagy-related gene 5 (ATG5) through the sequestration of microRNA-2355-5p, thereby promoting the development of DLBCL. Furthermore, research suggests that the combination of rituximab and chidamide might reduce treatment resistance by increasing the expression of autophagy-associated proteins (13). By specifically regulating the stability of carcinogenic signaling proteins, autophagy can also be involved in the biological processes of DLBCL. In the MCD subtype of DLBCL, characterized by MYD88L265P and CD79B mutations, TANK-binding kinase 1 (TBK1)-dependent selective autophagy can target and degrade the ubiquitinated MYD88L265P protein, and bruton tyrosine kinase (BTK) inhibitors appear to enhance this autophagic clearance, potentially leading to therapeutic benefits (14). All of these outcomes point to autophagy as a therapeutically useful and physiologically marked process in DLBCL.

A fundamental member of the ribosomal P protein family, ribosomal protein lateral stalk subunit P0 (RPLP0), is necessary for ribosome stability and translational elongation (15). There is growing evidence that numerous cancer types, such as lung and prostate cancer, exhibit abnormal expression of RPLP0 (16,17). The clinical prognosis and tumor progression are intimately linked to this imbalance. Reactive oxygen species (ROS) accumulate as a result of inhibition of RPLP family proteins, which initiates autophagy, reduces proliferation and induces G2 phase arrest, according to findings (18). These findings suggest that RPLP0-associated translational processes regulate autophagy and redox homeostasis in cancer cells. The PI3K/AKT/mTOR signaling cascade is a key oncogenic hub that integrates cellular stress, growth factor signals and nutrient availability (19). While PI3K/AKT/mTOR activation typically inhibits autophagic activity, pathway inhibition might increase autophagic flux and encourage cell death. The data presented suggest that RPLP0 may influence autophagy in DLBCL via the PI3K/AKT/mTOR pathway, with implications for biomarker development and patient stratification.

DLBCL may develop due to oxidative stress and altered mitochondrial activity, according to prior research (20,21). The autophagy system helps maintain cellular homeostasis under stressful conditions. Moreover, the AKT/mTOR signaling pathway is known to regulate cell survival and autophagy in response to environmental cues. The aim of the present study was to clarify the role of RPLP0 in regulating these cellular processes in DLBCL. Based on the aforementioned evidence, we hypothesized that RPLP0 promotes DLBCL cell survival by maintaining redox homeostasis and activating the AKT/mTOR pathway, thereby suppressing stress-induced autophagy. Investigating these pathways will shed light on the molecular causes of DLBCL and may reveal potential therapeutic targets.

Materials and methods

Data selection and differential expression analysis

The Gene Expression Omnibus Database (https://www.ncbi.nlm.nih.gov/gds/) and The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) provided the gene expression data for this present investigation. TCGA-DLBC dataset was selected, which includes 48 DLBCL tumor samples from TCGA and 929 normal control samples from the Genotype-Tissue Expression database (https://gtexportal.org/), and the GSE12453 dataset, containing 11 tumor samples and 25 normal samples (22). Differential expression analysis of genes in the two datasets was performed using the R package ‘Limma’ (version 3.54.2; Bioconductor; T Core Team) (23). Genes were screened based on fold change (FC), with FC>2 considered as upregulated differentially expressed genes (DEGs) and FC<0.5 considered as downregulated DEGs, with a significance criterion of P<0.05. Intersection analysis was then performed on the upregulated and downregulated DEGs to obtain key intersection genes utilizing Bioinformatics & Evolutionary Genomics (https://bioinformatics.psb.ugent.be/webtools/Venn/).

Protein-protein interaction (PPI) network and selection of hub genes

To assess the potential functional relevance of key intersecting genes, the authors analyzed the STRING database (https://string-db.org/) to construct a PPI network. The top 15 genes were further analyzed for their interaction networks using the Module Clustering Coefficient (MCC), Edge Percolated Component (EPC) and Maximum Neighborhood Component (MNC) algorithms in the CytoHubba plugin of Cytoscape software (version 3.10.1; Cytoscape Consortium). Subsequently, the top 15 genes from these three algorithms were subjected to intersection analysis to obtain candidate genes. Then, gene expression analysis of these candidate genes was performed on TCGA-DLBC and GSE12453 datasets using the Sanger website (http://vip.sangerbox.com/home.html). By analyzing gene expression levels in the normal and tumor groups, the present study identified the pivotal genes.

Cell culture

Human normal lymphoblastic GM12878 and DLBCL cell lines (OCI-LY7 and U2932) were acquired from Changsha Abiwei Biotechnology Co., Ltd. and the DLBCL cell line OCI-LY8 was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. OCI-LY8 cells were cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.). GM12878, OCI-LY7 and U2932 cells were cultured under the same conditions in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.). In all media, 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) were included. The cells were cultivated in an incubator with 5% CO2 at 37˚C for 24 h before subsequent experiments.

Cell transfection and treatment

Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell transfection in accordance with the manufacturer's recommendations. For RPLP0 knockdown studies, cells were transfected with three RPLP0-specific small interfering RNAs (siRNAs): si-RPLP0-1 (sense, 5'-GCUAAGGUUGAAGCCAAGGAA-3'; and antisense, 5'-UUCCUUGGCUUCAACCUUAGC-3'), si-RPLP0-2 (sense, 5'-UGAUCAAGACUGGAGACAAAG-3' and antisense, 5'-CUUUGUCUCCAGUCUUGAUC-3') and si-RPLP0-3 (sense, 5'-GCUGCUGAACAUGCUCAACAU-3' and antisense, 5'-AUGUUGAGCAUGUUCAGCCAGC-3') or a non-targeting control siRNA (si-NC; sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-ACGUGACACGUUCGGAGAATT-3') at a final concentration of 50 nM. For overexpression studies, cells were transfected with the corresponding empty vector or a pcDNA3.1(+)-RPLP0 expression plasmid generated using the pcDNA3.1(+) backbone (Invitrogen; Thermo Fisher Scientific, Inc.) with 2 µg plasmid DNA used per well in a 6-well plate. The plasmid construct was verified by DNA sequencing. For subsequent investigations, cells were harvested 48 h after transfection.

DLBCL cells require special treatment for autophagy studies. 3-Methyladenine (3-MA; Sigma-Aldrich; Merck KGaA) is an autophagy inhibitor that acts in the early stages of the autophagy pathway. To block autophagy, DLBCL cells were treated with 10 mM 3-MA for 48 h. Rapamycin (RAPA) is a typical mTOR inhibitor. DLBCL cells were treated with 25 nM RAPA (Sigma-Aldrich; Merck KGaA) for 24 h for further analysis. A traditional ROS scavenger, N-acetyl-L-cysteine (NAC; Sigma-Aldrich; Merck KGaA), lowers intracellular ROS levels by directly interacting with ROS through redox processes. To regulate ROS levels, DLBCL cells were treated with 5 mM NAC for 2 h before subsequent assays.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from OCI-LY7 and OCI-LY8 cells in the indicated treatment groups using TRIzol® reagent (Takara Bio, Inc.). Important procedures, including qPCR amplification, cDNA synthesis and RNA quality assessment, were carried out in accordance with the techniques outlined in earlier research (24). Specifically, cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Inc.). The reaction procedure included the removal of genomic DNA at 42˚C for 2 min, followed by reverse transcription at 37˚C for 15 min, and enzyme inactivation at 85˚C for 5 sec. qPCR amplification was performed using TB Green Premix Ex Taq II (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 5 sec and annealing/extension at 60˚C for 30 sec. Primer sequences are as follows: RPLP0: Forward: 5'-CGTCCTCGTGGAAGTGACAT-3', reverse: 5'-CTTGGAGCCCACATTGTCTG-3', GAPDH: forward: 5'-CATGTTGCAACCGGGAAGGA-3', reverse: 5'-ATCACCCGGAGGAGAAATCG-3'. mRNA expression was normalized using the endogenous reference GAPDH. The 2-ΔΔCq method was employed for relative quantification (25).

Western blotting (WB)

RIPA lysis buffer (Beyotime Biotechnology) containing phosphatase and protease inhibitors was used to extract total protein from cells. Following determination of protein content using a BCA Protein Assay Kit (Beyotime Biotechnology), in accordance with procedures described in previous studies, 30 µg of protein per lane was separated using 8-15% SDS-PAGE gels according to the molecular weights of the target proteins and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween-20 (TBST) (26). The membranes were incubated overnight with the primary antibody at 4˚C. The following are the primary antibodies utilized in this investigation: RPLP0 (cat. no. ab192866; 1:1,000), thioredoxin (TXN; cat. no. ab133524; 1:10,000), heat shock protein family A member 1A (HSPA1A; cat. no. ab5439; 1:1,000), heat shock protein family B member 1 (HSPB1; cat. no. ab2790; 1:1,000), microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A; cat. no. ab52628; 1:50,000), ATG5 (cat. no. ab108327; 1:1,000), cleaved caspase-3 (cat. no. ab32351; 1:2,000), caspase-3 (cat. no. ab184787; 1:2,000), phosphorylated (p)-AKT1 (cat. no. 66444-1-Ig; 1:2,000), AKT1 (cat. no. 10176-2-AP; 1:2,000), p-mTOR (cat. no. ab109268; 1:1,000), mTOR (cat. no. ab109268; 1:1,000) and GAPDH (cat. no. ab181602; 1:10,000). Among them, p-AKT1 and AKT1 antibody were derived from Wuhan Sanying Biotechnology. By contrast, the other primary antibodies were derived from Abcam. Following three washes with TBST containing 0.1% Tween-20, the membrane was incubated for 1 h at room temperature with goat anti-rabbit (cat. no. ab6721; 1:2,000; Abcam) and anti-mouse secondary antibodies (cat. no. ab6789; 1:2,000; Abcam) conjugated to horseradish peroxidase. Immobilon Western Chemiluminescent HRP Substrate (Merck KGaA) was used for chemiluminescent detection. Image Lab software (version 6.1; Bio-Rad Laboratories, Inc.). was installed to measure band intensity.

Colony formation assay

OCI-LY7 and OCI-LY8 cells were transfected with si-NC or si-RPLP0-1. Following transfection, cells in the logarithmic growth phase were collected, resuspended in complete culture medium, and seeded into 6-well plates at a density of 1x10³ cells per well. The cells were cultured at 37˚C in a humidified atmosphere containing 5% CO2 for 14 days, with the culture medium replaced every 3 days. After incubation, 4% paraformaldehyde was added to fix the cells for 15 min. After fixation, colonies were stained with nitroblue tetrazolium chloride for 30 min at room temperature. A cell cluster containing at least 50 cells was defined as a colony. Images were acquired using a chemiluminescence imaging system (Bio-Rad Laboratories, Inc.), and the colonies were counted using ImageJ software (version 1.53; National Institutes of Health).

Cell Counting Kit-8 (CCK-8) assay

96-well plates were seeded with DLBCL cells at a density of 1,000 cells per well. After cell collection, 10 µl CCK-8 reagent (Dojindo Laboratories, Inc.) was added to 100 µl culture medium. After 2 h incubation at 37˚C, an iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Inc.) was employed to quantify the optical density (OD) at 450 nm to identify cell growth.

Flow cytometry

As directed by the manufacturer, apoptosis was measured with an Annexin V-FITC/PI apoptosis detection kit (cat. no. ab14085; Abcam). Briefly, OCI-LY7 and OCI-LY8 cells were collected, cleaned in cold PBS and then reconstituted in 500 µl of 1X binding buffer. Annexin V-FITC (5 µl) was used to stain the cells at room temperature for 10 min in the dark, followed by PI (5 µl) for 5 min. Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Flow cytometric data were analyzed using FlowJo software (version 10.8.1; BD Biosciences).

For cell cycle analysis, cells were collected and fixed in pre-chilled 70% ethanol at -20˚C overnight. Following fixation, cells were washed with PBS, treated with RNase A (100 µg/ml), and stained with PI (50 µg/ml) for 30 min in the dark. A FACSCalibur flow cytometer was used to quantify DNA content, and the proportions of cells in different cell cycle phases were analyzed using FlowJo software (version 10.8.1; BD Biosciences).

Mitochondrial membrane potential (MMP) assay

A JC-1 staining kit (cat. no. ab113850; Abcam) was used to assess MMP in OCI-LY7 and OCI-LY8 cells according to the manufacturer's instructions. Cells were cultured with the JC-1 working solution for 20 min at 37˚C in the dark after the specified treatments. Following incubation, the cells were immediately examined under a fluorescence microscope (Nikon Corporation) after being cleaned with PBS. Red JC-1 aggregates indicate intact ΔΨm, whereas increased green JC-1 monomers reflect mitochondrial depolarization.

ROS level assay

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Biotechnology) was used to measure intracellular ROS levels in accordance with the manufacturer's directions. OCI-LY7 and OCI-LY8 cells were transfected with si-RPLP0-1 or si-NC, seeded on coverslips in 6-well plates, and incubated under standard culture conditions. Following the specified incubation period, cells were treated with DCFH-DA solution (10 µM) for 20 min in the dark at 37˚C after being rinsed with serum-free media. Excess probe was removed by washing three times with serum-free medium. DAPI (1 µg/ml) for 5 min was used to counterstain the nuclei, and a fluorescence microscope (Nikon Corporation) was used to capture fluorescence images rapidly.

Transmission electron microscope (TEM)

Cells with si-NC or si-RPLP0-1 were collected and fixed in 2.5% glutaraldehyde overnight at 4˚C. Following a phosphate buffer wash, cells were dehydrated by a graded ethanol series, post-fixed for 1 h with 1% osmium tetroxide, and then embedded in epoxy resin. The resin blocks were polymerized at 60˚C for 48 h before ultrathin sectioning. After cutting and mounting ultrathin slices (70 nm thick) on copper grids, they were sequentially stained with 2% uranyl acetate for 15 min and lead citrate for 10 min at room temperature. Mitochondrial morphology was observed with an HT7700 TEM (Hitachi Ltd.).

Determination of NAD+/NADH ratio

Intracellular NAD+/NADH ratios were measured using a commercial NAD+/NADH Assay Kit (cat. no. S0175, Beyotime Biotechnology) as directed by the manufacturer. Briefly, cells were harvested and lysed on ice in the NAD+/NADH extraction buffer provided in the kit. The supernatants were collected after centrifuging the lysates at 12,000 x g for 10 min at 4˚C. For total NAD (NAD+ + NADH) measurement, an aliquot of each extract was directly subjected to the enzymatic cycling reaction. To assess NADH, a parallel aliquot was chilled on ice after a 30 min incubation at 60°C to break down NAD+. Reaction mixtures were prepared according to the kit protocol, and absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad Laboratories, Inc.). NAD+ levels were assessed as total NAD minus NADH, and the NAD+/NADH ratio was computed as (total NAD-NADH)/NADH. Values were normalized to equal cell numbers (or total protein) across samples.

Statistical analysis

The R program (version 4.1.0; Posit Software, PBC) was used for the statistical analysis. Statistical significance was defined as P<0.05. Differences between groups were assessed using an unpaired Student's t-test (two groups), and one-way ANOVA with Tukey's post hoc test were employed to compare multiple groups. Each experiment was conducted at least three times, and the mean ± SD was used to describe the results.

Results

Identification and characterization of hub overlapping DEGs in DLBCL

In the present study, 6,338 upregulated and 1,115 downregulated DEGs were first identified from TCGA-DLBCL dataset (Fig. 1A). Additionally, from the GSE12453 dataset, 604 upregulated and 18 downregulated DEGs were detected (Fig. 1B). Venn diagram analysis was performed to identify overlapping DEGs between the two datasets, revealing 396 upregulated DEGs and 0 downregulated DEGs (Fig. 1C and D). PPI network analyses of these overlapping upregulated DEGs were conducted using the MCC, MNC and EPC algorithms. This analysis identified the top 15 most highly associated genes for each algorithm (Fig. 1E-G). Among these, six candidate genes (RPS11, RPL27, RPS18, RPS5, RPLP0 and RPS3) were selected as potential biomarkers (Fig. 1H). Expression analysis of these candidate genes in DLBCL-related datasets revealed that, compared with normal controls, all six genes exhibited notably higher expression in the tumor group (Fig. 1I and J). The present investigation examined RPLP0 because it is an essential component of the large ribosomal subunit and vital for protein synthesis.

Figure 1 Overlapping DEGs in DLBCL found in the GSE12453 and TCGA-DLBCL datasets. (A) A volcano plot shows the upregulated (orange) and downregulated (green) DEGs from TCGA-DLBCL dataset. (B) The volcano plot shows the upregulated (orange) and downregulated (green) DEGs found in the GSE12453 dataset. (C) Venn diagram showing the overlap of upregulated DEGs between TCGA-DLBCL and GSE12453 datasets. (D) Venn diagram illustrating the overlap of downregulated DEGs between the two datasets. PPI network analysis of the overlapping upregulated DEGs. The top 15 genes were selected using the (E) MCC, (F) MNC and (G) EPC algorithms. (H) The six candidate genes were identified from the MCC, MNC and EPC algorithms. (I and J) Expression of the six candidate genes (RPS11, RPL27, RPS18, RPS5, RPLP0 and RPS3) in DLBCL and normal groups in the (I) TCGA-DLBCL dataset and (J) GSE12453 datasets. ****P<0.0001. DLBCL, diffuse large B-cell lymphoma; DEGs, differentially expressed genes; PPI, protein-protein interaction; MCC, maximal clique centrality; MNC, maximum neighborhood component; EPC, edge percolated component; TCGA, The Cancer Genome Atlas.

Expression and knockdown efficiency of RPLP0 in DLBCL cell lines

The GM12878 cell line and DLBCL cell lines (OCI-LY7, OCI-LY8 and U2932) were subjected to RT-qPCR and WB analyses to assess RPLP0 levels in DLBCL. The results showed that RPLP0 expression was elevated in DLBCL cell lines, with OCI-LY7 and OCI-LY8 cells exhibiting the highest levels (Fig. 2A-C). Three distinct siRNA constructs targeting RPLP0 were transfected into OCI-LY7 and OCI-LY8 cells to investigate the function of RPLP0. RPLP0 knockdown markedly reduced RPLP0 mRNA and protein levels by RT-qPCR and WB analyses, with si-RPLP0-1 showing the most effective knockdown (Fig. 2D-H).

Figure 2 Expression and knockdown efficiency of RPLP0 in DLBCL cell lines. (A) RT-qPCR analysis of RPLP0 expression in the GM12878 (normal B cell), OCI-LY7, OCI-LY8 and U2932 cell lines. (B and C) WB analysis of RPLP0 expression in the GM12878 (normal B cell), OCI-LY7, OCI-LY8 and U2932 cell lines. Knockdown of RPLP0 using three different siRNA constructs (si-RPLP0-1, si-RPLP0-2 and si-RPLP0-3) in (D) OCI-LY7 and (E) OCI-LY8 cells. The efficiency of RPLP0 knockdown was assessed by RT-qPCR. The x-axis represents the siRNA constructs used (si-RPLP0-1, si-RPLP0-2 and si-RPLP0-3), and the y-axis shows relative mRNA levels. (F) The efficiency of RPLP0 knockdown was assessed by WB. The y-axis shows relative protein levels in (G) OCI-LY7 and (H) OCI-LY8. *P<0.05, **P<0.01 and ***P<0.001. DLBCL, diffuse large B-cell lymphoma; RT-qPCR, reverse transcription-quantitative PCR; WB, western blot; si, small interfering RNA; NC, negative control.

RPLP0 knockdown disrupts proliferation, cell cycle and apoptosis in DLBCL cell lines

Colony formation assays assessed the effects of RPLP0 knockdown on cell proliferation. The findings showed that, compared with the control group, silencing RPLP0 reduced the ability of OCI-LY7 and OCI-LY8 cells to proliferate (Fig. 3A and B). The effects of RPLP0 knockdown on the cell cycle distribution of OCI-LY7 and OCI-LY8 cells were next evaluated by flow cytometry. The outcomes revealed a concurrent build-up of cells in the G2 phase and a notable decline in the proportion of cells in the S phase (Fig. 3C and D). Additionally, apoptosis experiments were conducted to determine if RPLP0 knockdown affected cell death. However, there were no appreciable variations in the rates of apoptosis between the si-NC control group and the RPLP0 knockdown group in DLBCL cell lines (Fig. 3E and F). These outcomes imply that RPLP0 mainly controls DLBCL cell proliferation through cell cycle modulation.

Figure 3 Effects of RPLP0 knockdown on cell proliferation, cell cycle distribution and apoptosis in OCI-LY7 and OCI-LY8 cell lines. (A) Colony formation assay assessing the proliferative capacity of OCI-LY7 and OCI-LY8 cells following RPLP0 knockdown. (B) The x-axis represents the different groups (si-NC vs. si-RPLP0-1) and the y-axis shows the number of colonies formed. (C) Cell cycle analysis by flow cytometry in OCI-LY7 and OCI-LY8 cells after RPLP0 knockdown. (D) The x-axis represents the cell cycle phases (G1, S and G2), and the y-axis represents the percentage of cells in each phase. (E) Apoptosis analysis by flow cytometry in OCI-LY7 and OCI-LY8 cells after RPLP0 knockdown. (F) Quantification of the apoptosis rate in the indicated groups (si-NC, si-RPLP0-1). **P<0.01. ns, not significant; si, small interfering RNA; NC, negative control; RPLP0, ribosomal protein lateral stalk subunit P0.

RPLP0 knockdown disrupts redox homeostasis and induces mitochondrial dysfunction in DLBCL cells

The impact of RPLP0 knockdown on oxidative damage repair markers in DLBCL cell lines was further examined. According to WB analysis, si-RPLP0-1 markedly decreased TXN, HSPA1A and HSPB1 protein expression compared with the control group (Fig. 4A-C). This is supported by the notable increase in the NAD+/NADH ratio following RPLP0 knockdown (Fig. 4D). Next, the authors utilized JC-1 staining to measure MMPs. Cells with silenced RPLP0 showed decreased JC-1 aggregates and increased JC-1 monomers, indicating mitochondrial depolarization. Quantitative analysis of the red/green fluorescence ratio further confirmed a marked reduction in membrane potential compared with the si-NC group (Fig. 4E-F). Additionally, the si-RPLP0-1 group exhibited greater green fluorescence intensity in DCFH-DA staining, and quantitative analysis of ROS fluorescence intensity revealed a marked increase, suggesting enhanced intracellular ROS accumulation (Fig. 4G-H). These outcomes imply that RPLP0 deficiency worsens mitochondrial function and oxidative damage in DLBCL cells.

Figure 4 Effects of RPLP0 knockdown on oxidative stress and mitochondrial function in OCI-LY7 and OCI-LY8 cell lines. (A) Representative western blotting images showing the expression of TXN, HSPA1A and HSPB1 in OCI-LY7 and OCI-LY8 cells following RPLP0 knockdown. GAPDH was used as the loading control. (B) Quantitative analysis of TXN, HSPA1A and HSPB1 protein expression in OCI-LY7 cells. (C) Quantitative analysis of TXN, HSPA1A and HSPB1 protein expression in OCI-LY8 cells. (D) Measurement of the NAD+/NADH ratio in OCI-LY7 and OCI-LY8 cells following si-RPLP0-1 transfection. The x-axis represents the treatment groups (si-NC vs. si-RPLP0-1) and the y-axis shows the NAD+/NADH ratio. (E) Mitochondrial membrane potential analysis by JC-1 staining in RPLP0-knockdown OCI-LY7 and OCI-LY8 cell. Scale bar: 50 µm. (F) Quantitative analysis of the JC-1 red/green fluorescence ratio. (G) ROS levels in OCI-LY7 and OCI-LY8 cells following si-RPLP0-1 transfection, measured by 7'-dichlorodihydrofluorescein diacetate fluorescence. Scale bar, 50 µm. (H) Quantitative analysis of relative reactive oxygen species fluorescence intensity. *P<0.05, **P<0.01 and ***P<0.001. ROS, reactive oxygen species; si, small interfering RNA; NC, negative control; RPLP0, ribosomal protein lateral stalk subunit P0; TXN, thioredoxin; HSPA1A, heat shock protein family A member 1A; HSPB1, heat shock protein family B member 1.

RPLP0 deletion alters autophagy regulation and mitochondrial morphology in DLBCL cells

The transformation of LC3-I into LC3-II is a crucial stage in the creation of autophagosomes. In OCI-LY7 and OCI-LY8 cells transfected with si-RPLP0-1, the protein levels of LC3 and ATG5 were measured using WB to investigate the effect of RPLP0 knockdown on autophagy. The present findings demonstrated that RPLP0 knockdown increased the LC3-II/I ratio and upregulated ATG5 protein expression in both cell lines (Fig. 5A-C). TEM was subsequently used to examine the mitochondrial morphology of RPLP0-silenced cells. The findings revealed notable signs of autophagy, including an increase in the number of autophagic vacuoles in the field of view and quantification confirmed a notable increase in the number of autolysosomes per field (Fig. 5D and E). Next, the autophagy inhibitor 3-MA (5 mM) was added to RPLP0-silenced OCI-LY7 and OCI-LY8 cells. According to WB analysis, RPLP0 knockdown increased the LC3-II/I ratio but had no discernible effect on caspase-3 expression. Nevertheless, 3-MA treatment reversed the effects of RPLP0 knockdown, obviously reducing the LC3-II/I ratio and upregulating cleaved caspase-3 expression (Fig. 5F-H). The present findings suggest that RPLP0 knockdown increases autophagy in DLBCL cells by modulating ATG5 and LC3-II expression.

Figure 5 Impact of RPLP0 knockdown on autophagy markers, mitochondrial morphology and the effects of 3-MA treatment in OCI-LY7 and OCI-LY8 cells. (A) WB analysis of autophagy-related proteins LC3 and ATG5 in OCI-LY7 and OCI-LY8 cells transfected with si-RPLP0-1. (B) Quantitative analysis of the LC3-II/LC3-I ratio in OCI-LY7 and OCI-LY8 cells. (C) Quantitative analysis of ATG5 protein expression in OCI-LY7 and OCI-LY8 cells. (D) Transmission electron microscopy images showing increased autophagic vacuoles in RPLP0-silenced cells compared with controls, with (E) quantitative analysis of the number of autolysosomes per field. Scale bar, 2 µm. (F) WB analysis of LC3-I, LC3-II, cleaved Caspase-3 and Caspase-3 expression in OCI-LY7 and OCI-LY8 cells treated with 3-MA (5 mM) following si-RPLP0-1 transfection. (G) Quantitative analysis of the LC3-II/LC3-I ratio in the indicated treatment groups. (H) Quantitative analysis of Caspase-3 protein expression in the indicated treatment groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and ns, not significant. WB, western blotting; 3-MA, 3-methyladenine; RPLP0, ribosomal protein lateral stalk subunit P0; si, small interfering RNA; NC, negative control; ATG5, autophagy-related gene 5.

Overexpression of RPLP0 activates AKT/mTOR signaling and inhibits autophagy in DLBCL cells

The transfection efficiency of the RPLP0 overexpression plasmid was evaluated via RT-qPCR and WB in subsequent experiments. Investigations demonstrated that levels of RPLP0 mRNA and protein expression of both cell lines were markedly increased after RPLP0 overexpression (Fig. 6A-C). The effects of RPLP0 overexpression on total and phosphorylated AKT and mTOR were examined by WB analysis. The present findings indicated that although there were no appreciable changes in the overall expression levels of AKT and mTOR, RPLP0 overexpression increased the levels of p-AKT and p-mTOR (Fig. 6D-F). According to these data, RPLP0 may trigger the AKT/mTOR pathway in DLBCL cells. The impact of RPLP0 overexpression and the mTOR inhibitor RAPA (25 nM) on LC3 and ATG5 expression in OCI-LY7 and OCI-LY8 cells was further investigated. The present results demonstrated that RPLP0 overexpression reduced the LC3-II/I ratio and decreased ATG5 levels. However, co-treatment with RAPA partially reversed these effects, markedly restoring the LC3-II/I ratio and ATG5 protein levels (Fig. 6G-I). These results indicate that RPLP0 overexpression activates the AKT/mTOR pathway, thereby inhibiting stress-induced autophagy in DLBCL cells.

Figure 6 Effects of RPLP0 overexpression on the AKT/mTOR pathway and autophagy in OCI-LY7 and OCI-LY8 cell lines. (A) RT-qPCR analysis of RPLP0 expression in OCI-LY7 and OCI-LY8 cells transfected with the RPLP0 overexpression plasmid. The x-axis represents the treatment groups, and the y-axis shows relative mRNA expression levels. (B) WB analysis of RPLP0 expression in OCI-LY7 and OCI-LY8 cells transfected with the RPLP0 overexpression plasmid. (C) Quantitative analysis of RPLP0 protein expression in OCI-LY7 and OCI-LY8 cells. (D) Representative WB images showing the expression of phosphorylated AKT, total AKT, phosphorylated mTOR and total mTOR in OCI-LY7 and OCI-LY8 cells following RPLP0 overexpression. GAPDH was used as the loading control. (E) Quantitative analysis of the phosphorylated AKT/total AKT ratio. (F) Quantitative analysis of the phosphorylated mTOR/total mTOR ratio. (G) Representative WB images showing LC3-I, LC3-II and ATG5 expression in OCI-LY7 and OCI-LY8 cells following RPLP0 overexpression, with or without RAPA treatment (25 nM). GAPDH was used as the loading control. (H) Quantitative analysis of the LC3-II/LC3-I ratio in the indicated treatment groups. (I) Quantitative analysis of ATG5 protein expression in the indicated treatment groups. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; RAPA; rapamycin, WB, western blotting; ATG5, autophagy-related gene 5.

RPLP0 modulates ROS-induced AKT/mTOR signaling and autophagy in DLBCL cells

NAC is a well-known ROS scavenger that specifically reduces intracellular ROS levels by directly reacting with ROS in redox reactions. To explore the role of RPLP0 in ROS regulation and its impact on downstream signaling, the authors investigated the effects of RPLP0 knockdown and NAC treatment (5 mM) on the AKT/mTOR signaling pathway in OCI-LY7 and OCI-LY8 cell lines. RPLP0 knockdown notably decreased p-AKT and p-mTOR, according to WB analysis. However, upon NAC treatment, the levels of p-AKT and p-mTOR were restored to control levels, while the total protein expression of AKT and mTOR remained unaffected (Fig. 7A-C). The WB findings then demonstrated that RPLP0 knockdown increased the LC3-II/I ratio and ATG5 expression. Following NAC therapy, ATG5 expression returned to baseline, and the LC3-II/I ratio normalized (Fig. 7D-F). Additionally, the CCK-8 assay was employed to measure cell viability. The present results showed that NAC therapy could restore the considerable decline in cell viability caused by RPLP0 knockdown (Fig. 7G and H). According to these findings, RPLP0 controls the buildup of ROS in DLBCL, thereby activating the AKT/mTOR signaling pathway and preventing oxidative stress-induced autophagy.

Figure 7 Effects of RPLP0 knockdown and NAC treatment on AKT/mTOR signaling, autophagy, and cell viability in DLBCL cell lines. (A) Representative WB images and quantitative analysis of AKT/mTOR pathway-related proteins in OCI-LY7 and OCI-LY8 cells after RPLP0 knockdown, with or without NAC treatment (5 mM). (B) Quantitative analysis of the phosphorylated AKT/total AKT ratio in the indicated treatment groups. (C) Quantitative analysis of the phosphorylated mTOR/total mTOR ratio in the indicated treatment groups. (D) Representative WB images and quantitative analysis of LC3 and autophagy-related gene 5 expression in OCI-LY7 and OCI-LY8 cells after RPLP0 knockdown, with or without NAC treatment. (E) Quantitative analysis of the LC3-II/LC3-I ratio in the indicated treatment groups. (F) Quantitative analysis of autophagy-related protein 5 expression in the indicated treatment groups. (G and H) Cell viability analysis using CCK-8 assays in (G) OCI-LY7 and (H) OCI-LY8 cells following RPLP0 knockdown and NAC treatment. The x-axis represents the treatment groups, and the y-axis shows relative cell viability. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. DLBCL, diffuse large B-cell lymphoma; WB, western blot; NAC, N-acetyl-L-cysteine; CCK-8, Cell Counting Kit-8; RPLP0, ribosomal protein lateral stalk subunit P0; si, small interfering RNA; NC, negative control; p-, phosphorylated.

Discussion

By combining TCGA-DLBCL and GSE12453 datasets, 396 overlapping upregulated genes were found. Six ribosome-related candidate genes (RPS11, RPL27, RPS18, RPS5, RPLP0 and RPS3) were found to be markedly differentially expressed in DLBCL based on PPI network analysis. Experiments on cellular function revealed that RPLP0 knockdown reduced cell survival and colony-forming capacity. Concurrently, RPLP0 knockdown altered the cell cycle distribution, decreasing S phase and increasing G2 phase accumulation, whereas apoptosis levels remained constant. Mechanistically, RPLP0 knockdown caused mitochondrial depolarization, elevated intracellular ROS levels and disrupted redox equilibrium. RPLP0 knockdown caused autophagy (LC3-II accumulation and ATG5 overexpression) and decreased the levels of oxidative stress-related proteins (TXN, HSPA1A and HSPB1). Conversely, RPLP0 overexpression reduced autophagy markers and increased AKT/mTOR phosphorylation, which RAPA partially reversed. Importantly, scavenging ROS with NAC restored AKT/mTOR activity, normalized autophagy-related proteins and rescued the viability of RPLP0-silenced cells. These results indicate that RPLP0 promotes DLBCL cell survival by coordinating the ROS-dependent AKT/mTOR signaling pathway to limit stress-induced autophagy.

DLBCL is a highly aggressive malignant tumor characterized by rapid proliferation and immense biosynthetic demands; therefore, ribosome biogenesis and protein translation are important for maintaining malignant growth (27). Consistent with this, ribosomal gene signatures have shown prognostic value in DLBCL, providing a basis for risk stratification and potential therapeutic targets. A previous study supports the functional relevance of ribosomal proteins in the biology of DLBCL. RPS6 is upregulated in DLBCL, and its knockdown suppresses proliferation (28). Low-dose RAPA modulates 5'TOP mRNA translation, a process regulated by RPS6, linking RPS6 to aberrant protein synthesis in DLBCL. In the present study, the authors integrated DLBCL-related datasets and identified six consistently upregulated ribosome-related candidate genes (RPS11, RPL27, RPS18, RPS5, RPLP0 and RPS3). Although these genes have not yet been fully studied in DLBCL, cumulative evidence from other malignancies highlights their pro-tumorigenic potential. For example, aberrant upregulation of RPS3 has been linked to cancer progression and treatment resistance through the NF-κB pathway, while RPL27 has been reported to promote colorectal cancer progression; its depletion impairs cell cycle progression and cell proliferation and attenuates the malignant phenotype (29,30). Notably, RPLP0 is also associated with different cancers, including cervical cancer, breast cancer, lung adenocarcinoma and gastric cancer, with high expression correlating with tumorigenesis and poor prognosis (31-33). A mechanistic study suggests that the upstream tumor suppressor phospholipase A and acyltransferase 4 can regulate RPLP0 to inhibit proliferation and survival, and may regulate key cell cycle checkpoints through CDK2 and p21-related pathways (34). Consistent with these observations, the present data show that RPLP0 is notably upregulated in DLBCL cells, and its knockdown inhibits proliferation and induces G2 phase arrest, supporting RPLP0 as a potentially clinically relevant functional hub gene in DLBCL.

ROS serve a regulatory role in cellular activity, but excessive levels can lead to oxidative stress, which damages cellular structure and function (35). Modulating ROS may be a potential treatment approach for DLBCL, as the loss of mitochondrial membrane potential during apoptosis is frequently accompanied by elevated ROS levels (36). To preserve intracellular redox homeostasis, TXN is a crucial redox-regulating protein (37). Classic indicators of heat shock and protein toxicity stress, HSPA1A and HSPB1, can prevent irreversible protein aggregation under stressful conditions (38,39). According to Feng et al (40) DCZ0358 suppresses cell growth and induces G0/G1 cell cycle arrest in DLBCL by impairing mitochondrial function and increasing ROS levels. The JNK signaling pathway is then triggered, encouraging apoptosis. Additionally, prior research has demonstrated that downregulation of ribosomal P complex proteins (such as RPLP0, RPLP1 or RPLP2) results in the buildup of ROS, which in turn causes endoplasmic reticulum stress and the unfolded protein response, ultimately triggering autophagy (41). According to research by Meng et al (42) RPLP0 is overexpressed in hepatocellular carcinoma (HCC) and is associated with a bad prognosis. RPLP0 regulates the ROS-JAK2/STAT3-c-Myc pathway, forming a positive feedback loop with c-Myc to promote HCC progression. Consistent with these observations, the present data demonstrate that RPLP0 knockdown in DLBCL downregulates TXN, HSPA1A and HSPB1, thereby altering the cellular redox state and elevating the NAD+/NADH ratio. Additionally, RPLP0 knockdown causes mitochondrial depolarization, which eventually results in a large build-up of ROS. Notably, despite these pro-apoptotic signals, apoptosis remained unchanged, which the authors attribute to concurrent activation of protective autophagy, which buffers against cell death, as supported by 3-MA and NAC experiments. In conclusion, the present results imply that RPLP0 serves an essential role in maintaining redox homeostasis and mitochondrial integrity in DLBCL cells and may be a potential therapeutic target.

A number of studies have shown that autophagy reduces oxidative stress by eliminating damaged mitochondria, thereby averting oxidative stress-induced cellular damage (43,44). According to Zhang et al (45) patients with DLBCL had considerably higher levels of the autophagy marker p62 than those with reactive lymphoid hyperplasia, and higher p62 expression was associated with a shorter 2-year progression-free survival. Chen et al (46) demonstrated that artesunate (ART) induces autophagy, ferroptosis, apoptosis and cell cycle arrest in DLBCL cells by blocking the STAT3 signaling pathway. Furthermore, ART-induced apoptosis was mitigated by suppression of autophagy and ferroptosis, underscoring the complex interactions between these cellular mechanisms. To engage in autophagy, LC3, a crucial protein on the autophagosome membrane, changes from LC3-I to LC3-II (47). ATG5 promotes the development and growth of the autophagosomal membrane by interacting with ATG12 and ATG16L1(48). In the present investigation, RPLP0 knockdown in DLBCL cells increased ATG5 expression and elevated the LC3-II/I ratio. TEM revealed an increased number of autophagosomes, suggesting activation of autophagy. Furthermore, treatment with the autophagy inhibitor 3-MA markedly reduced the LC3-II/I ratio. These outcomes suggest that RPLP0 deficiency may control autophagy, which may help DLBCL cells manage oxidative stress and mitochondrial malfunction.

The PI3K/AKT/mTOR signaling pathway is a well-known regulatory system for autophagy that is key for several biological processes (49). Studies have shown that the loss of ribosomal proteins reduces mTOR phosphorylation in cancer cells, thereby affecting autophagy and the regulation of proliferation (50,51). According to research by Wang et al (52) research, macrophage-capping protein is upregulated in DLBCL and activates the PI3K/AKT signaling pathway to promote cell invasion and proliferation. Similarly, Li et al (53) noted that in pancreatic ductal adenocarcinoma, ROS activate AMPK to trigger autophagy while inhibiting mTORC1. The authors' research demonstrates that tailored selective autophagy can reduce the risk of adverse effects. Du et al (54) found that plumbagin (PL) increased ROS generation and inhibited the growth of DLBCL cells. Additionally, PL decreased the expression of p-Akt, p-PI3K, Bcl-2 and p-mTOR while increasing the expression of cleaved Caspase-3 and Bax. These findings suggest that PL induces apoptosis in DLBCL cells by triggering oxidative stress, which in turn inactivates the PI3K/AKT/mTOR signal pathway. In the present study, RPLP0 overexpression was shown to activate the AKT/mTOR signaling pathway, thereby suppressing autophagy and increasing cell survival. Conversely, RPLP0 knockdown increases autophagy, suppresses AKT/mTOR phosphorylation and causes ROS buildup in DLBCL cells. It is noteworthy that NAC can reverse alterations in autophagy and restore AKT/mTOR function. These findings emphasize the therapeutic promise of RPLP0, revealing its critical role in regulating cell survival in DLBCL by orchestrating the autophagy process, thereby modulating the AKT/mTOR signaling pathway.

Several limitations of the present study should be acknowledged. First, all functional experiments were performed only in DLBCL cell lines, without in vivo validation using animal models. Second, the present findings are primarily derived from cell-based assays and public database analyses and lack direct validation with independent clinical DLBCL specimens. Third, the present study did not investigate whether RPLP0 exerts differential effects across other DLBCL subtypes, as only germinal center B-cell-like-subtype cell lines were used. Future studies will establish DLBCL xenograft mouse models, validate RPLP0 expression and its clinical relevance using larger patient cohorts, and explore its subtype-specific roles using additional cell lines and clinical samples from different molecular subtypes.

In conclusion, the present research examined the vital role of RPLP0 in controlling autophagy, mitochondrial activity and oxidative stress in DLBCL. Functional studies demonstrated that RPLP0 knockdown led to G2 cell cycle arrest and reduced cell growth. By controlling mitochondrial function, RPLP0 knockdown also made oxidative stress worse in DLBCL. Additionally, RPLP0 overexpression can stimulate the AKT/mTOR signaling pathway and suppress autophagy in DLBCL cells, whereas RPLP0 deficiency can enhance autophagy. Notably, by triggering the ROS-induced AKT/mTOR signaling pathway, RPLP0 markedly contributes to the survival of DLBCL cells under oxidative stress. These discoveries may guide the development of therapies targeting the RPLP0-related pathway and deepen understanding of DLBCL.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

Conception and design of the research was performed by SW, XY and JT. Acquisition of data was performed by SW and XY. Analysis and interpretation of data was the responsibility of SW, XY, RM, BH, DS and NM. RM, BH, SS and NM performed the statistical analysis. SW and XY drafted the manuscript. JT revised the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript. SW and XY confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

During the preparation of this work, artificial intelligence tools were used to improve the readability and language of the manuscript or to generate images, and subsequently, the authors revised and edited the content produced by the artificial intelligence tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

References