MicroRNA-129-5p Regulates Glycolysis and Cell Proliferation by Targeting the Glucose Transporter SLC2A3 in Gastric Cancer Cells

Abstract

Tumor cells increase their glucose consumption through aerobic glycolysis to manufacture the necessary biomass required for proliferation, commonly known as the Warburg effect. Accumulating evidences suggest that microRNAs (miRNAs) interact with their target genes and contribute to metabolic reprogramming in cancer cells. By integrating high-throughput screening data and the existing miRNA expression datasets, we explored the roles of candidate glycometabolism-regulating miRNAs in gastric cancer (GC). Subsequent investigation of the characterized miRNAs indicated that miR-129-5p inhibits glucose metabolism in GC cells. miRNA-129-5p directly targets the 3'-UTR of SLC2A3, thereby suppressing glucose consumption, lactate production, cellular ATP levels, and glucose uptake of GC cells. In addition, the PI3K-Akt and MAPK signaling pathways are involved in the effects of the miR-129-5p/SLC2A3 axis, regulating GC glucose metabolism and growth. These results reveal a novel role of the miR-129-5p/SLC2A3 axis in reprogramming the glycometabolism process in GC cells and indicate a potential therapeutic target for the treatment of this disease.

Keywords: SLC2A3; cancer metabolism; gastric cancer; miR-129-5p; proliferation.

FIGURE 1

MicroRNA-129-5p is a repressor of glucose metabolism in GC cells. (A) A schematic diagram of the protocol used to search for candidate metabolism-associated miRNAs in GC. (B) Eight miRNAs were identified as candidate glycometabolism-regulating miRNAs in GC. (C,D) Lactate production in MGC-803 cells after transfection of the indicated miRNAs (C) and corresponding inhibitors (D). (E) Lactate production, glucose consumption and cellular ATP levels in GC cells transfected with miR-129-5p mimic. Values are shown as the mean ± standard error of the mean (SEM), n = 3 in (C–E). ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

FIGURE 2

SLC2A3 is the direct target of miR-129-5p in GC cells. (A) Schematic representation of the strategy used to identify candidate target genes of miR-129-5p. (B) Diagram of putative miR-129-5p binding sites in the 3′-UTR of SLC2A3. The mutant sequences of SLC2A3 3′-UTR used in the luciferase reporter constructs are indicated in red. (C) Relative activities of luciferase reporters containing SLC2A3 3′-UTR variants co-transfected with miR-129-5p or negative control mimics in HEK 293T cells. (D) SLC2A3 mRNA and protein levels in GC cells transfected with miR-129-5p mimics. Values are shown as the mean ± SEM, n = 3 in (C,D). ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 3

The miR-129-5p/SLC2A3 axis regulates glucose metabolism in GC cells. (A) SLC2A3 knockdown suppressed lactate production, glucose consumption, cellular ATP levels and glucose uptake in GC cells. (B) The restoration of SLC2A3 protein expression in GC cells significantly abolished the suppressive effects of miR-129-5p on lactate excretion, glucose consumption, cellular ATP levels and glucose uptake in GC cells. Values are shown as the mean ± SEM, n = 3. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 4

The miR-129-5p/SLC2A3 axis regulates the proliferation of GC cells. (A) miR-129-5p suppressed DNA synthesis in GC cells. Upper: Representative images of EdU assays (the scale represents 200 μm, original magnification × 200). Lower: Quantification of the EdU incorporation rate in GC cells. (B) miR-129-5p mimics inhibited GC cell colony formation. Upper: Representative images. Lower: Quantification of colony numbers. (C) The reintroduction of SLC2A3 significantly reversed miR-129-5p-induced inhibition of cell colony formation. Upper: Representative images. Lower: Quantification of colony numbers. Values are shown as the mean ± SEM, n = 3. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 5

miR-129-5p reprograms gene expression profiling in GC cells. (A) Functional gene annotation clustering of genes regulated by miR-129-5p in MGC-803 cells. Significantly enriched groups are ranked based on the group enrichment scores, suggested by the gene ontology terms. Green, signaling pathway. Blue, biological process. (B) Expression levels of the subsetting genes involved in PI3K-Akt signaling pathway and MAPK signaling pathway. The genes are shaded with blue or red in the heatmap to indicate low or high expression, respectively. (C) qPCR analysis for the selected genes from ranked pathways of MGC-803 cells transfected with miR-129-5p mimics or negative controls. (D) Western blotting assays for PI3K-Akt and MAPK signaling pathways in SGC-7901 and MGC-803 cells transfected with miR-129-5p mimics or SLC2A3 siRNAs. (E) CCK8 assays for SGC-7901 and MGC-803 cells transfected with miR-129-5p mimics, with or without PDGF-BB treatment. Values are shown as the mean ± SEM, n = 3 in (C,E). ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns, not significant. β-actin served as internal control.