SUCLG1 promotes aerobic respiration and progression in plexiform neurofibroma

Abstract

Plexiform neurofibromas (PNFs) are benign tumors that affect 20‑50% of patients with type I neurofibromatosis (NF1). PNF carries a risk of malignancy. There is no effective cure for PNF. Its onset may be associated with genetic and metabolic abnormalities, but the exact mechanisms remain unclear. Succinate‑CoA ligase GDP/ADP‑Forming Subunit α(SUCLG1), a catalytic enzyme in the tricarboxylic acid cycle, is highly expressed in PNF. The present study aimed to explore the role of SUCLG1 in function and metabolism of PNF cells. SUCLG1 expression was verified using western blotting and immunofluorescence. After inducing SUCLG1 knockdown and overexpression, functional changes in PNF cells were assessed, as well as effects of SUCLG1 on cell respiration and glucose metabolism. Quantitative PCR, WB, electron microscopy and Flow cytometry demonstrated that SUCLG1 enhanced mitochondrial quality and promoted mitochondrial fusion, thereby driving proliferation and migration of tumor cells, inhibiting apoptosis and altering the cell cycle. A Seahorse assay showed that elevated SUCLG1 expression enhanced cell aerobic respiration without affecting the glycolytic process. This suggests that SUCLG1 upregulation in PNF does not trigger the Warburg effect associated with malignant tumors. This study also demonstrated the positive regulation of cellular function by promoting the expression level of the SLC25A1 gene when SUCLG1 expression was elevated. In conclusion, SUCLG1 altered the mechanism of mitochondrial quality control to enhance cell aerobic respiration, thereby driving the pathogenesis of PNF. Thus, SUCLG1 can serve as a potential target in future therapeutic strategies.

Keywords: aerobic respiration; mitochondria; neurofibromatosis type 1; plexiform neurofibroma; tricarboxylic acid cycle.

Figure 1

Analysis of proteomics and metabolomics. (A) Distribution of pathways in which differentially expressed proteins and metabolites were enriched. (B) Proteins and metabolites enriched in the TCA. SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; K01899, succinate-CoA ligase GDP-forming subunit α; K01902, succinate-CoA ligase ADP-forming subunit α; TCA, tricarboxylic acid cycle.

Figure 2

SUCLG1 and CA are highly expressed in PNF. (A) Representative immunofluorescence staining of (B) SUCLG1 expression in normal skin and PNF. Magnification, x50x. (C) Western blotting for (D) SUCLG1 expression in ipNF95.6, ipNF05.5 and HSCs. (E) CA content in ipNF95.6, ipNF05.5 and HSCs. ^*P<0.05, ^**P<0.01, ^***P<0.001. SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; CA, citric acid; PNF, plexiform neurofibroma; HSC, human Schwann cell; NFM, neurofilament medium.

Figure 3

Regulation of PNF cell function by SUCLG1 expression. (A) Reverse transcription-quantitative PCR analysis of SUCLG1 in ipNF05.5 and ipNF95.6 cells after transfection. (B) Western blotting of (C) SUCLG1 expression in ipNF05.5 and ipNF95.6 after transfection. Proliferation of (D) ipNF95.6 and (E) ipNF05.5 cells. (F) Wound healing assay showing, Magnification, x100x. (G) Wound width of PNF cells after transfection. (H) Representative flow cytometry of (I) cell cycle distribution in PNF cells after transfection. (J) Representative flow cytometry of (K) apoptosis in PNF cells after transfection. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. 0 h. ns, not significant; PNF, plexiform neurofibroma; SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; sh, short hairpin; NC, negative control; OE, overexpression; OD, optical density.

Figure 4

Effect of SUCLG1 on intracellular metabolism and mitochondrial fusion. (A) OCR. (B) Changes in proton leak and ATP production in mitochondrial respiration. (C) Changes in basal respiration and spare respiratory capacity in mitochondrial respiration. (D) Glycolysis stress test. (E) Parameters of glycolytic function. (F) Mitochondrial mass was estimated by ratio of mtDNA to gDNA. (G) Representative transmission electron microscopy images of mitochondria. Magnification: 500x. Arrows indicate elongated mitochondria. (H) Representative western blotting of mitochondrial fusion proteins OPA1, MFN1 and MFN2. (I) ROS concentration. ^*P<0.05, ^**P<0.01, ^***P<0.001. ns, not significant; OCR, oxygen consumption rate; ECAR, extracelluar acidification rate; SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; ROS, reactive oxygen species; mtDNA, mitochondrial DNA; OPA, Optic Atrophy Protein ; MFN, mitochondrial fusion protein; sh, short hairpin; NC, negative control; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; DG, deoxy-glucose; OE, overexpression.

Figure 5

Effect of SUCLG1 expression on SLC25A1. (A) Representative Western blotting for (B) ACLY, (C) SLC25A1 and (D) CS. ELISA determination of (E) CS and (F) ACLY protein activity. (G) Cell proliferation was evaluated by the Cell Counting Kit-8 assay. (H) Wound healing assay. Magnification, x100. (I) Wound width of ipNF95.6 after transfection with NC and OE-SUCLG1 viruses and addition of SLC25A1 inhibitor. (J) Flow cytometry of (K) cell cycle distribution following transfection with NC and OE-SUCLG1 viruses and adding SLC25A1 inhibitor. (L) Flow cytometry of (M) apoptosis following transfection and adding SLC25A1 inhibitor. ^&&&P<0.001 vs. OE-SUCLG1 inhibitor. ^***P<0.001 vs. NC, ^###P<0.001 vs. OE-SUCLG1, ^*P<0.05 vs. NC. SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; ns, no significance; OD, optical density; SLC25A1, ;Solute Carrier Family 25 Member 1; ACLY, ATP Citrate Lyase; CS, Citrate Synthase; NC, negative control; OE, overexpression; sh, short hairpin.