m6A RNA methylation-mediated NDUFA4 promotes cell proliferation and metabolism in gastric cancer

Abstract

Gastric cancer (GC) is a malignancy with poor prognosis. NDUFA4 is reported to correlate with the progression of GC. However, its underlying mechanism in GC is unknown. Our study was to reveal the pathogenic mechanism of NDUFA4 in GC. NDUFA4 expression was explored in single-cell and bulk RNA-seq data as well as GC tissue microarray. Mitochondrial respiration and glycolysis were estimated by oxygen consumption rate and extracellular acidification rate, respectively. The interaction between NDUFA4 and METTL3 was validated by RNA immunoprecipitation. Flow cytometry was used to estimate cell cycle, apoptosis and mitochondrial activities. NDUFA4 was highly expressed in GC and its high expression indicated a poor prognosis. The knockdown of NDUFA4 could reduce cell proliferation and inhibit tumor growth. Meanwhile, NDUFA4 could promote glycolytic and oxidative metabolism in GC cells, whereas the inhibition of glycolysis suppressed the proliferation and tumor growth of GC. Besides, NDUFA4 inhibited ROS level and promoted MMP level in GC cells, whereas the inhibition of mitochondrial fission could reverse NDUFA4-induced glycolytic and oxidative metabolism and tumor growth of GC. Additionally, METTL3 could increase the m6A level of NDUFA4 mRNA via the m6A reader IGF2BP1 to promote NDUFA4 expression in GC cells. Our study revealed that NDUFA4 was increased by m6A methylation and could promote GC development via enhancing cell glycolysis and mitochondrial fission. NDUFA4 was a potential target for GC treatment.

Fig. 1. NDUFA4 was up-regulated in GC and its elevation indicated poor prognosis.

A Uniform Manifold Approximation and Projection (UMAP) plot of patients with non-atrophic gastritis (NAG), chronic atrophic gastritis (CAG), wild intestinal metaplasia (IMW), severe intestinal metaplasia (IMS) and EGC in GSE134520 are denoted by distinct colors. B UMAP plot showed the distribution of epithelial cells in patients with NAG, CAG, IMW, IMS and EGC, marked by the expression of marker gene epithelial cell adhesion molecule (EPCAM) (yellow, high; purple, low). C Differentially expressed genes in epithelial cells between patients with EGC and NAG (the significantly expressed gene, NDUFA4, is highlighted). D UMAP plot of NDUFA4 expression in patients with NAG, CAG, IMW, IMS and EGC (yellow, high; purple, low). E NDUFA4 expressionin epithelial cells between patients with EGC and NAG. F, G NDUFA4 expression in paired gastric and adjacent normal tissues in hospital cohort (F) and GSE33335 dataset (G). H Survival analysis and comparison among people with high and low values of NDUFA4 expression in GSE22377 database. I Representative IHC images and (J) scores of NDUFA4 in GC tissue microarrays. Scale bar: 100 μm. K Survival analysis and comparison among people with high and low expression of NDUFA4 expression in GC tissue microarrays. L The relative mRNA and protein levels of NDUFA4 in various GC cells and normal human gastric epithelium. *P < 0.05, ***P < 0.001 vs adjacent-normal or GES-1.

Fig. 2. NDUFA4 promoted cell proliferation and reduced cell cycle arrest and apoptosis in GC cells.

A Cell viability, (B, C) colony formation and (D, E) cell cycleof AGS, HGC27 and MKN45 cells with or without NDUFA4 overexpression or knockdown. *P < 0.05, **P < 0.01, ***P < 0.001 vs shNC or vector.

Fig. 3. NDUFA4 promoted GC growth in vivo.

AGS cells transduced with shNC or NDUFA4 shRNA vector and MKN45 cells transduced with NDUFA4 expression vector were subcutaneously injected into the armpits of the nude mice, respectively. A, D Tumor volume in each group. B, E Tumor weight in each group. C, F Expression of NDUFA4, Cyclin D1 and CDK4 in each group. ***P < 0.001 vs. shNC or vector.

Fig. 4. NDUFA4 promotes glycolytic and oxidative metabolism in GC cells.

A ECAR, (B) OCR, (C) lactate, (D) ATP content and (E) expression of ENO1 and LDHA in AGS, HGC27 and MKN45 cells with or without NDUFA4 overexpression or knockdown. ***P < 0.001 vs shNC or vector.

Fig. 5. Inhibition of glycolysis exerted anti-tumor effects against GC.

A Cell viability, (B) colony formation and (C, D) cell cycle of AGS and MKN45 cells treated with 10 mM 2-DG or vehicle. AGS or MKN45 cells were subcutaneously injected into the mice with or without 100 mg/kg 2-DG treatment. E Tumor volume was estimated. F The photograph of tumors. G Tumor weight in each group. **P < 0.01, ***P < 0.001.

Fig. 6. NDUFA4 regulated the level of ROS and MMP in GC cells.

(A, B) ROS, (C, D) MMP and (E) expression of OPA1, p-Drp1 and PGC1α in AGS and MKN45 cells modulated as indicated. ***P < 0.001 vs shNC or vector.

Fig. 7. Inhibition of mitochondrial fission reversed NDUFA4-mediated effects in GC.

A Cell viability, (B) ECAR, (C) OCR, (D) lactate and (E) ATP content of MKN45 cells with NDUFA4 overexpression and/or 20 μM Mdivi-1. MKN45 cells transduced with NDUFA4 expression vector were subcutaneously injected into the armpits of the mice with or without 25 mg/kg Mdivi-1 treatment. F Tumor volume in each group. G The photograph of xenografts. H Tumor weight in each group. ***P < 0.001.

Fig. 8. METTL3 promoted the m6A modification of NDUFA4 via the m6A reader IGF2BP1.

A NDUFA4 expression in AGS cells treated with 100 μM 3-Deazaadenosine (DAA) for 0, 12, 24, and 48 h. B–F AGS cells were transfected with METTL14 siRNA. The whole m6A level (B), m6A level of NDUFA4 3’UTR (C), and abundance of NDUFA4 3’UTR (D) were detected. The expression of METTL3 (E) and NDUFA4 (F) was detected. G–H IGF2BP1 expression (G) and NDUFA4 mRNA stability (H) in AGS cells transfected with IGF2BP1 shRNA. I The interaction between IGF2BP1 and NDUFA4 3′UTR was detected by RIP assay. ***P < 0.001 vs 0 h, siNC, shNC or IgG.