关键词: 脂肪间充质干细胞, 外泌体, miR-126-3p, 人脐静脉内皮细胞, 糖脂毒性, AKT通路

Abstract: BACKGROUND: MiR-126-3p from adipose-derived mesenchymal stem cells released exosomes (AMSC-Exos) has been used as a potential biomarker of diabetes, but few studies of glucolipotoxicity and mechanism of miR-126-3p on human umbilical vein endothelial cells are found.  
OBJECTIVE: To explore the effect and mechanism of miR-126-3p from AMSC-Exos on glucolipotoxicity of human umbilical vein endothelial cells.
METHODS:  (1) Glucolipotoxicity model of human umbilical vein endothelial cells was induced by 30 mmol/L glucose and 100 µmol/L palmitic acid in vitro. (2) The supernatant of passage 4 AMSC-Exos was collected. The morphology of AMSC-Exos was observed by transmission electron microscope. The size distribution of AMSC-Exos was determined by nanoparticle tracking analysis. (3) Human umbilical vein endothelial cells with glucolipotoxicity were treated with AMSC-Exos. The content of reactive oxygen species and apoptosis rate were detected by flow cytometry. ELISA was implemented to detect the expression of inflammatory factors. (4) RT-qPCR was used to detect the expression levels of miR-126-3p mRNA in AMSC-Exos and normal human umbilical vein endothelial cells co-cultured with AMSC. (5) miR-126-3p overexpression was transfected into human umbilical vein endothelial cells. Cell proliferation was detected by CCK-8 assay. Western blot assay was used to detect the expression levels of apoptotic proteins. (6) The target genes of miR-126-3p were predicted and verified by bioinformatics software. Dual luciferase reporter gene assay was applied to verify and predict signaling pathway. (7) CRK overexpression was transfected into human umbilical vein endothelial cells with glucolipotoxicity. The expression levels of apoptotic protein and p-AKT were detected by western blot assay. (8) Human umbilical vein endothelial cell glucolipotoxicity model was co-treated with AKT pathway inhibitor RG7440 and miR-126-3p overexpression/AMSC-Exos. Apoptotic protein expression was detected by western blot assay and apoptosis rate was detected by flow cytometry.  
RESULTS AND CONCLUSION: (1) AMSC-Exos were round or oval membranous vesicles, and the particle size ranged from 30 to 200 nm. (2) Compared with the model group, AMSC-Exos could remarkably reduce the expression levels of interleukin-1β, interleukin-6 and interleukin-8 (P < 0.05, P < 0.001), and obviously reduced the reactive oxygen species and cell apoptosis rate (P < 0.05). (3) Compared with adipose-derived mesenchymal stem cells, mRNA expression level of miR-126-3p from AMSC-Exos was significantly increased (P < 0.001). Compared with human umbilical vein endothelial cells, co-culture with adipose-derived mesenchymal stem cells could significantly increase the expression level of miR-126-3p of human umbilical vein endothelial cells (P < 0.001). (4) miR-126-3p overexpression could significantly promote cell proliferation, reduce cell apoptotic rate and expression levels of cleaved caspase 3, 9 (P < 0.01, P < 0.001). (5) Bioinformatics analysis and dual luciferase reporter gene assay proved that CRK was the target gene of miR-126-3p. The AKT pathway was as the object of next experimental research. (6) CRK overexpression could activate AKT pathway and up-regulate expression level of cleaved caspase 3, 9. Compared with the Exos group, the cell apoptosis rate was significantly higher in the Exos + AKT inhibitor group (P < 0.001). (7) The results showed that miR-126-3p from AMSC-Exos could improve the glucolipotoxicity of human umbilical vein endothelial cells. It is supposed that the mechanism maybe work by miR-126-3p regulating the AKT pathway via targeting CRK gene.

Key words: adipose-derived mesenchymal stem cells, exosomes, miR-126-6p, human umbilical vein endothelial cells, glucolipotoxicity, AKT pathway