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. 2020 Mar 30;13(1):46-54.
doi: 10.15283/ijsc19088.

miR-450a-5p Eliminates MGO-Induced Insulin Resistance via Targeting CREB

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Free PMC article

miR-450a-5p Eliminates MGO-Induced Insulin Resistance via Targeting CREB

Cuifeng Wei et al. Int J Stem Cells. .
Free PMC article

Abstract

Background and objectives: miR-450a-5p was involved in fat formation, however, its role in insulin resistance remains unclear. This study investigated the effects of miR-450a-5p on endothelial cells, with the aim of finding a potential target for diabetes mellitus.

Methods and results: Human umbilical vein endothelial cells (HUVECs) were treated with low-glucose, high-glucose, methylglyoxal (MGO), and insulin alone or in combination with MGO. The expression of miR-450a-5p in treated cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity, migration and fat formation were determined by MTT experiments, Transwell assay and oil red O staining. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by double-luciferase reporter analysis, and its effects on eNOS/AKT pathway were estimated. We found that the expression of miR-450a-5p was decreased obviously in endothelial cells treated with high-glucose and MGO. In vitro cell experiments showed that MGO could not only promote the activity of endothelial cells, but also accelerate cell migration and fat accumulation, which, however, could be reversed by up-regulation of miR-450a-5p. Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin, and such effects were reversed by up-regulation of miR-450a-5p. Furthermore, CREB was the target gene for miR-450a-5p, had an activation effect on the eNOS/AKT pathway.

Conclusions: Up-regulated miR-450a-5p eliminates MGO-induced insulin resistance via targeting CREB, and therefore could be used as a potential target to improve insulin resistance and treat patients with diabetes-related diseases.

Keywords: High-glucose; Insulin resistance; Methylglyoxal; eNOS/AKT pathway; miR-450a-5p.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Methylglyoxal (MGO) down-regulated the expression of miR-450a-5p in human umbilical vein endothelial cells (HUVECs). (A) Quantitative real-time polymerase chain reaction (qRT-PCR) assay measured the expression of miR-450a-5p in HUVECs after treatment with low-glucose (5 mM), high-glucose (25 mM), or low-glucose combined with xylose (4.5 mM) for 24, 48, 72 h. (B) qRT-PCR assay determined the expression of miR-450a-5p in HUVECs treated by different concentrations of MGO (0, 250, 500, 1000 μM). (C) qRT-PCR assay determined the expression of miR-450a-5p in HUVECs treated by control, MGO only or combined with aminoguanidine bicarbonate salt (AG). **p<0.001, vs. low-glucose, 0 μm, or Control; ##p<0.001, vs. MGO. n=3.
Fig. 2
Fig. 2
Over-expressed miR-450a-5p inhibited viability, migration, and lipid formation of methylglyoxal (MGO)-induced human umbilical vein endothelial cells (HUVECs). Mimic control, miR-450a-5p mimic, inhibitor control, and miR-450a-5p inhibitor were respectively transfected into MGO-treated HUVECs. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) assay measured the expression of miR-450a-5p in MGO-treated HUVECs after the transfection. (B) MTT assay detected the optical densities (OD) values of MGO-induced HUVECs after the transfection for 24, 48 and 72 h. (C) Quantitative analysis of Transwell assay in MGO-treated HUVECs after the transfection. (D) Microscopic images of Transwell and Oil red O staining experiments in MGO-induced HUVECs after transfection. ^^p<0.001, vs. Control; **p<0.001, vs. MGO+mimic control; ##p<0.001, vs. MGO+inhibitor control. n=3.
Fig. 3
Fig. 3
Over-expressed miR-450a-5p inhibited the insulin resistance of methylglyoxal (MGO)-induced human umbilical vein endothelial cells (HUVECs). In this figure, HUVECs were respectively treated by control, insulin (Ins), MGO, insulin combined with MGO, miR-450a-5p mimic plus MGO, or mimic plus MGO and insulin. (A∼C) Western blot (WB) bands and quantitative analysis determined the expressions of eNOS/AKT pathway-related proteins in HUVECs. (D) Griess reaction assessed the release of NO in HUVECs. ^^p<0.001, vs. Control; **p<0.001, vs. Ins; ##p<0.001, vs. Ins+MGO. n=3.
Fig. 4
Fig. 4
CREB was the target gene for miR-450a-5p. (A) Targetscan7.2 predicted that the position 108-114 of CREB1 3’UTR was the binding site of miR-450a-5p. (B) Double-luciferase reporter analysis measured the luciferase activity of human umbilical vein endothelial cells (HUVECs) after transfection with miR-450a-5p mimic plus wild type CREB (CREB-WT) or its mutant type (CREB-MUT). (C) Quantitative real-time polymerase chain reaction (qRT-PCR) assay measured the expression of CREB in methylglyoxal (MGO)-induced HUVECs after transfection with mimic control, miR-450a-5p mimic, mimic plus negative control (NC) or CREB. ^^p<0.001, vs. Blank, or Control; **p<0.001, vs. MGO+mimic control; ##p<0.001, vs. MGO+mimic+NC. n=3.
Fig. 5
Fig. 5
Over-expressed miR-450a-5p reduced the insulin resistance of methylglyoxal (MGO)-induced human umbilical vein endothelial cells (HUVECs) by targeting CREB. Western blot (WB) bands and quantitative analysis determined the expressions of eNOS/AKT pathway-related proteins in MGO-induced HUVECs after treatment with control, insulin (Ins), MGO, MGO+Ins, MGO+miR-450a-5p mimic+negative control (NC), MGO+Ins+mimic+NC, MGO+mimic+CREB, or MGO+Ins+mimic+CREB. ^^p<0.001, vs. Control; **p<0.001, vs. MGO+mimic+NC; ##p<0.001, vs. MGO+mimic+CREB. n=3.

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