To explore the role of miR-148/152 family in the regulation of glycolysis-related genes in pluripotent stem cell-derived endothelial cells (ECs) .

In this study, we used the wild-type human embryonic stem cells (hESCs) strain H1 (WT) and the miR-148/152 family knockout hESCs line (TKO) , which was generated from H1 hESCs using CRISPR/Cas9 gene editing technology. The expression of pluripotent marker NANOG was detected by immunofluorescence technology. ECs were differentiated from both WT and TKO hESCs by subsequent treatment with chemical small molecules and cytokines such as Wnt signaling pathway activator, basic fibroblast growth factor, vascular endothelial growth factor and bone morphogenetic protein 4. RT-qPCR was performed to detect the knockout efficiency of miR-148/152 in the TKO ECs, and explore the effects of miR-148/152 family on the glycolytic metabolic enzymes and the metabolic transformation-related genes. Data between the two groups were compared by t test.

Compared with the WT, the TKO hESCs showed significantly decreased detection abundance of miR-148a (1.00±0.03 vs 0.00±0.00) , miR- 148b (1.00±0.07 vs 0.13±0.06) , and miR-152 (1.01±0.15 vs 0.05±0.03) , (all P < 0.001) . A comparable expression of the pluripotency marker NANOG was detected in the nuclei of WT and TKO hESCs. Both WT and TKO hESCs can differentiate into CD31-positive ECs. Compared with the WT, the ECs derived from TKO hESCs showed significantly decreased detection abundance of miR-148a (1.00±0.05 vs 0.00±0.00) , miR-148b (1.00±0.08 vs 0.12±0.05) , and miR- 152 (1.00±0.08 vs 0.13±0.07) , (all P < 0.001) . The mRNA expression levels of key glycolytic enzymes, including phosphoglycerate kinase 1 (1.00±0.09 vs 0.20±0.02) , hexokinase 2 (1.02±0.20 vs 0.55±0.12) , 6-phosphofructo-2-kinase (1.00±0.05 vs 0.67±0.14) , lactate dehydrogenase A (1.00±0.04 vs 0.53±0.05) , pyruvate kinase M (1.00±0.03 vs 0.83±0.09) , glyceraldehyde-3-phosphate dehydrogenase (1.00±0.03 vs 0.59±0.09) , were significantly reduced in TKO hESC-derived ECs, when compared to that in the WT hESC-derived ECs (P < 0.05) . Moreover, the mRNA expression of pyruvate dehydrogenase kinase 1 (1.00±0.08 vs 2.90±0.23, P < 0.001) , a key gene in the process of metabolic transformation from glucose metabolism to oxidative phosphorylation metabolism, was significantly upregulated in TKO hESC-derived ECs. Consistently, expression level of the glycolysis suppressor phosphate and tension homology in the TKO ECs increased by about 4 folds when compared to that in the WT ECs (1.01±0.11 vs 3.83±0.81, P < 0.001) .

The miR-148/152 family is an important factor in regulating the glucose metabolism of ECs, and may contribute to maintaining the balance of glycolysis and inhibiting the glycolysis-to-oxidative phosphorylation transition.