MRPL35 Induces Proliferation, Invasion, and Glutamine Metabolism in NSCLC Cells by Upregulating SLC7A5 Expression

Abstract

Background: Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non-small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear.

Methods: The detection of mRNA and protein of MRPL35, ubiquitin-specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT-PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α-ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis.

Results: MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells.

Conclusion: MRPL35 expression was stabilized by USP39-induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

Keywords: MRPL35; SLC7A5; USP39; deubiquitination; glutamine metabolism.

FIGURE 1

MRPL35 is highly expressed in NSCLC tissues and cell lines. (A–C) TCGA, CPTAC, and ENCORI databases showed the high expression of MRPL35 in lung cancer tissues. (D) The Kaplan–Meier plotter database suggested that high MRPL35 expression was associated with shorter survival times in NSCLC patients. (E) IHC analysis for MRPL35 expression in NSCLC tissues and normal tissues. (F) qRT‐PCR analysis for MRPL35 mRNA expression in NSCLC tissues and normal tissues. (G) Kaplan–Meier overall survival curves for 37 NSCLC patients classified according to relative MRPL35 expression level. (H, I) Western blotting analysis for MRPL35 protein expression in NSCLC tissues and normal tissues, as well as in NSCLC cell lines and normal 16HBE cells. *p < 0.05.

FIGURE 2

MRPL35 knockdown suppresses NSCLC cell proliferation, invasion, and glutamine metabolism and induces cell apoptosis. (A–H) H1299 and A549 cells were transfected with si‐MRPL35 or si‐NC. (A) Western blotting analysis for MRPL35 protein expression in cells. (B, C) Measurement of cell proliferation by MTT and EdU assays. (D) Flow cytometry for cell apoptosis. (E) Transwell assay for cell invasion analysis. (F–H) Glutamine metabolism analysis by detecting glutamine consumption and the production of α‐ketoglutarate and glutamate. *p < 0.05.

FIGURE 3

USP39 induces MRPL35 deubiquitination and stabilizes its expression. (A) Western blotting analysis for MRPL35 protein expression in H1299 and A549 cells after incubating with PR‐619 (2.5 or 5 μm). (B) The effects of USP enzyme families on MRPL35 protein expression in H1299 and A549 cells were investigated by western blotting. (C) The transfection efficiencies of si‐USP39, USP39, or the control (si‐NC or pcDNA) were verified by detecting USP39 levels by western blotting in NSCLC cells. (D, E) qRT‐PCR and western blotting analyses for MRPL35 protein expression in NSCLC cells after USP39 silencing or overexpression. (F) NSCLC cells were transfected with si‐NC, si‐USP39, or si‐USP39 + MG132, and MRPL35 protein expression was measured by western blotting. (G) Western blotting for MRPL35 protein in NSCLC cells after USP39 overexpression under CHX treatment. (H) IP and western blotting assays detected ubiquitin‐MRPL35 levels in 293 T cells with indicated antibodies. *p < 0.05.

FIGURE 4

USP39 knockdown suppresses NSCLC cell proliferation, invasion, and glutamine metabolism and induces cell apoptosis by MRPL35. (A–I) H1299 and A549 cells were transfected with si‐USP39 or si‐USP39 and MRPL35. (A) Western blotting analysis for MRPL35 protein expression in cells. (B, C) Measurement of cell proliferation by MTT and EdU assays. (D, E) Flow cytometry for cell apoptosis. (F, G) Transwell assay for cell invasion analysis. (H–J) Glutamine metabolism analysis by detecting glutamine consumption and the production of α‐ketoglutarate and glutamate. *p < 0.05.

FIGURE 5

SLC7A5 expression is higher in NSCLC tissues and cells and is affected by MRPL35. (A) Heat map of deregulated genes in A549 cells after MRPL35 knockdown according to the GSE225959 dataset. (B) Protein levels of SLC7A5 in H1299 and A549 cells after MRPL35 knockdown. (C–E) TCGA, CPTAC, and ENCORI databases showed the high expression of SLC7A5 in lung cancer tissues. (F) qRT‐PCR analysis for SLC7A5 in NSCLC tissues and normal tissues. (G) Correlation analysis between SLC7A5 and MRPL35 mRNA in NSCLC tissues. (H) Western blotting analysis for SLC7A5 protein expression in NSCLC cell lines and normal 16HBE cells. *p < 0.05.

FIGURE 6

MRPL35 knockdown suppresses NSCLC cell proliferation, invasion, and glutamine metabolism and induces cell apoptosis by regulating SLC7A5. (A–I) H1299 and A549 cells were transfected with si‐MRPL35 or si‐MRPL35 and SLC7A5. (A) Western blotting analysis for SLC7A5 protein expression in cells. (B, C) Measurement of cell proliferation by MTT and EdU assays. (D, E) Flow cytometry for cell apoptosis. (F, G) Transwell assay for cell invasion analysis. (H–J) Glutamine metabolism analysis by detecting glutamine consumption and the production of α‐ketoglutarate and glutamate. *p < 0.05.

FIGURE 7

MRPL35 knockdown impedes NSCLC tumor growth in vivo. (A) Tumor volume of mice in each group. (B) Representative xenografts and tumor weight in each group. (C) Western blotting analysis for SLC7A5 protein expression in xenografts of each group. (D) IHC staining for Ki67, MMP9, and SLC7A5 protein in xenografts of each group. *p < 0.05.