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

  • Authors:
    • Shanshan Wang
    • Xutao Yang
    • Rochelle Mcgowan
    • Belinda Hodge
    • Daniel Shan
    • Nicolas Miller
    • Jiahong Tang
  • View Affiliations / Copyright

    Affiliations: Department of Hematology and Oncology, Hospital of Joint Logistics Support Force, Zhengzhou, Henan 450007, P.R. China, Department of Genetics, DICAT National Biomedical Computation Centre, Vancouver BC V6B 5A6, Canada
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 269
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    Published online on: August 7, 2026
       https://doi.org/10.3892/etm.2026.13264
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Abstract

Diffuse large B‑cell lymphoma (DLBCL) is a common, aggressive subtype of non‑Hodgkin lymphoma with poor outcomes. Identifying the primary molecular causes of DLBCL remains key. The present study examined the function of ribosomal protein lateral stalk subunit P0 (RPLP0) in DLBCL pathogenesis. The Cancer Genome Atlas‑DLBCL and GSE12453 datasets overlapping differentially expressed genes were identified. Hub genes were identified via protein‑protein interaction network analysis. DLBCL cells were subjected to functional tests following RPLP0 overexpression or knockdown. Reverse transcription‑quantitative PCR, western blotting, flow cytometry, transmission electron microscopy, colony formation assay and biochemical analysis were among the tests performed. N‑acetylcysteine (NAC), rapamycin (RAPA) and 3‑MA were among the medication therapies. In the DLBCL datasets, six ribosome‑associated genes were differentially expressed. RPLP0 knockdown inhibited the proliferation of DLBCL cells and caused G2‑phase arrest, without impacting apoptosis. Thioredoxin, heat shock protein family A member 1A and heat shock protein family B member 1 expression was downregulated by RPLP0 knockdown, which also increased the NAD+/NADH ratio, promoted reactive oxygen species (ROS) accumulation and caused mitochondrial membrane potential depolarization. Meanwhile, 3‑MA reversed the effects of RPLP0 knockdown, which encouraged LC3‑II accumulation, autophagy‑related gene 5 (ATG5) overexpression and an increase in autophagic vesicles. Autophagy‑related indicators were decreased, and AKT/mTOR phosphorylation was increased by RPLP0 overexpression, which RAPA inhibited. NAC therapy preserved the viability of RPLP0‑silenced cells, restored p‑AKT/p‑mTOR levels and restored normal LC3 and ATG5 expression. These findings suggest that RPLP0 regulates stress‑induced autophagy through ROS‑dependent AKT/mTOR signaling and may represent a potential therapeutic target for DLBCL.
View Figures

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.

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.

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.

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.

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.

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.

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.
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Copy and paste a formatted citation
Spandidos Publications style
Wang S, Yang X, Mcgowan R, Hodge B, Shan D, Miller N and Tang J: <em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy. Exp Ther Med 32: 269, 2026.
APA
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., & Tang, J. (2026). <em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy. Experimental and Therapeutic Medicine, 32, 269. https://doi.org/10.3892/etm.2026.13264
MLA
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., Tang, J."<em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy". Experimental and Therapeutic Medicine 32.4 (2026): 269.
Chicago
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., Tang, J."<em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy". Experimental and Therapeutic Medicine 32, no. 4 (2026): 269. https://doi.org/10.3892/etm.2026.13264
Copy and paste a formatted citation
x
Spandidos Publications style
Wang S, Yang X, Mcgowan R, Hodge B, Shan D, Miller N and Tang J: <em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy. Exp Ther Med 32: 269, 2026.
APA
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., & Tang, J. (2026). <em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy. Experimental and Therapeutic Medicine, 32, 269. https://doi.org/10.3892/etm.2026.13264
MLA
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., Tang, J."<em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy". Experimental and Therapeutic Medicine 32.4 (2026): 269.
Chicago
Wang, S., Yang, X., Mcgowan, R., Hodge, B., Shan, D., Miller, N., Tang, J."<em>RPLP0</em> drives diffuse large B‑cell lymphoma cell proliferation through reactive oxygen species‑dependent AKT/mTOR activation and inhibition of stress‑induced autophagy". Experimental and Therapeutic Medicine 32, no. 4 (2026): 269. https://doi.org/10.3892/etm.2026.13264
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