miR-210 in Exosomes Derived from Macrophages under High Glucose Promotes Mouse Diabetic Obesity Pathogenesis by Suppressing NDUFA4 Expression

Figure 2

Suppression of adipocyte glucose uptake, CIV activity, and NDUFA4 expression by macrophage-derived exosomes. (a) Glucose uptake by 3T3-L1 cells suppressed by macrophage-derived exosomes under high glucose treatment. Glucose content in 3T3-L1 cells was analyzed by the fluorometric method. (b) Mitochondrial respiratory chain complex IV activity in 3T3-L1 cells treated with exosomes derived from macrophages under high glucose treatment. ELISA was performed to analyze CIV activity. (c, d) Expression levels of miR-210 (c) and NDUFA4 gene (d) in 3T3-L1 cells treated with exosomes derived from macrophages. miR-210 expression and NDUFA4 expression were determined by quantitative RT-PCR. (e) NDUFA4 protein levels in 3T3-L1 cells treated with exosomes derived from macrophages by western blotting. VDAC1 was used as the internal standard. The 3T3-L1 cells cultured under normal conditions were used as the control group in above assays. (f) The predicted binding of miR-210 with 3-UTR regions of NDUFA4 gene. Targeting NDUFA4 gene by miR-210 in 3T3-L1 cells confirmed by dual-luciferase reporter assay. (g) The possible interaction between miR-210 and NDUFA4 was examined by pull-down assay using bio-miR-210 probes, and the relative expression of NDUFA4 was quantified. NG-exo: normal glucose exosomes; HG-exo: high glucose exosomes; CIV: complex IV; NDUFA4: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4; VDAC1: voltage-dependent anion channel 1; WT: wild type; MUT: mutant; NC: negative control; (compared with the normal glucose exosome group); (compared with the normal glucose exosome or NDUFA4-WT+NC group); (compared with the input group); ^### (compared with the bio-NC probe group).

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