RPL35 promotes neuroblastoma progression via the enhanced aerobic glycolysis

Abstract

Neuroblastoma (NB) is an rare type of tumor that almost affects children age 5 or younger due to its rapid proliferation ability. The overall survival rate of patients with advanced NB is not satisfactory. Ribosomal proteins (RPs) play a critical role in the development and progress of cancer. However, the contribution of RPL35 in NB has not been proven. In this study, we reveal that RPL35 is upregulated in NB tissues and the upregulation of RPL35 promotes proliferation and migration of NB while RPL35 knockdown significantly restrained the proliferation of NB cells. In terms of mechanism, glycolysis was decreased and the mitochondrial respiration was increased with knockdown of RPL35 in NB cells, indicating that RPL35 function as a positive regulator in aerobic glycolysis. Importantly, our data indicated that RPL35 deficiency decreased HIF1α expression both in mRNA and protein levels. Western blot analysis showed that RPL35 knockdown has a negative regulatory effect on the ERK pathway, and RPL35 modulated aerobic glycolysis in part through its regulation of the RPL35/ERK/HIF1α axis. Overall, RPL35 functions as a positive regulator of aerobic glycolysis, and the RPL35/ERK/HIF1α axis could be a potential therapeutic target for the therapy of NB.

Keywords: ERK; HIF1α; Neuroblastoma; RPL35; aerobic glycolysis.

Figure 1

RPL35 is upregulated in human neuroblastoma. A. Immunohistochemistry showing RPL35 expression in examples of NB tumor patient samples and normal tissue from different clinical samples. B. Kaplan-Meier curve showing overall survival of 28 NB patients with high or low RPL35 expression. C. Western blot analysis of RPL35 in NB tissue and normal tissue from different clinical samples. D, E. The mRNA level of RPL35 in NB tissue and normal tissue from different clinical samples. F. The protein level of RPL35 in different NB cell lines (DG, SH-SY5Y, NB-1643, NB-1691, SK-N-BE(2), BE(2)-C, SK-N-AS and IMR32). *P<0.05, **P<0.01.

Figure 2

RPL35 promotes the proliferation and migration of NB. A. The cell viability of SH-SY5Y and BE(2)-C at different times (0 h, 24 h, 48 h, 72 h, and 96 h) after infection with Lenti-RPL35 or Lenti-vector were evaluated by CCK8. B. The cloning formation of SH-SY5Y and BE(2)-C cells after infected with Lenti-RPL35 or Lenti-vector. C, D. The cell invasive ability measured by transwell assay. E. The migration potential evaluated by the wound healing assay. Scale bar =100 μm. *P<0.05, **P<0.01.

Figure 3

Knockdown of RPL35 impairs proliferation and migration of NB cells. A. The effect of RPL35 knockdown on SH-SY5Y and BE(2)-C cells viability was detected by CCK-8 assay at the indicated time. B. The effect of RPL35 knockdown on the cloning formation of SH-SY5Y and BE(2)-C cells. C. The effect of RPL35 knockdown on migration ability and invasion ability was determined after shRNA transfection in SH-SY5Y and BE(2)-C cells using a migration assay and a transwell assay. D. The migration potential of SH-SY5Y and BE(2)-C cells treated with shNC and shRPL35 were evaluated by the wound healing test. Scale bar =100 μm. *P<0.05, **P<0.01.

Figure 4

Knockdown of RPL35 leads to suppression of NB progression in vivo. A. Quantitative analysis of the NB tumor volume at the indicated time. B. Quantitative analysis of the NB tumor weight. C. HE staining, Ki-67, and CD34 immunohistological staining of tumors from shNC and shRPL35 cells. Scale bar =50 μm. D. HE staining for lung occupancy of shRPL35 or shNC expressing NB cells. Scale bar =100 μm. E. The survival rate of nude-mice after NB cell implantation. *P<0.05, **P<0.01.

Figure 5

Downregulation of RPL35 suppresses glycolysis of NB cells in vitro. (A, B) Diagram and quantitative of ECAR results obtained by Seahorse extracellular flux analyzer to determine the impact of shRPL35 on aerobic glycolysis in SH-SY5Y and BE(2)-C cells. (C, D) Diagram and quantitative of OCR measurement with Seahorse analyzer to confirm the role of shRPL35 in mitochondrial respiration. (E, F) The proliferation of NB cells that stably knockdown (E) or overexpressed (F) of RPL35 by the administration of the glycolytic inhibitor 2-DG (2 mM) measured by CCK8. *P<0.05, **P<0.01.

Figure 6

shRPL35 downregulates glycolytic proteins HK2 and LDHB. A, B. The Q-PCR analysis of GLUT1, HK2, LDHA, LDHB, and PDK1 mRNA level in shRPL35 and control SH-SY5Y and BE(2)-C cells. C. The protein levels of HK2, LDHB, and HIF1α in different NB tissues. D. The mRNA levels of HK2, LDHB in different NB tissues. *P<0.05, **P<0.01.

Figure 7

RPL35 regulates HIF1α transcriptional activity through Erk activation in NB. A. HIF1α transcriptional activity in NB cells overexpressed with different doses of RPL35 measured with the dual-luciferase assay. B. Western blot analysis of ERK phosphorylation as well as the HIF1α expression in RPL35 knockdown NB cells. C. ERK2-mutant mostly abolished the inhibition effect of shRPL35 on HIF1α transcriptional activity in NB cells. D. Diagram and quantitative of ECAR results obtained by Seahorse extracellular flux analyzer to determine the impact of ERK-mutant to shRPL35’s effect on aerobic glycolysis in SH-SY5Y and BE(2)-C cells. *P<0.05, **P<0.01 vs. shNC. #P<0.05, ##P<0.01 vs. shRPL35.