J F Bao1, J Hao, J Liu, W J Yuan, Q Yu. 1. Department of Nephrology, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, P. R. China. yuqingsl0618@163.com.
Abstract
OBJECTIVE: To determine the expression level of the microRNA in the process of epithelial-mesenchymal transition (EMT) of the peritoneal mesothelial cells (PMCs) induced by high glucose. MATERIALS AND METHODS: The PMCs were cultured using M199 medium with 10% fetal bovine serum, and the EMT was induced by D-glucose stimulation. Epithelial-mesenchymal transition was determined by changes in cell morphology and the expression levels of the EMT marker genes. Changes in cell morphology were observed by inverted microscope, and the expression levels of the EMT marker genes were determined by real-time PCR. The expression levels of the microRNA were detected by real-time PCR with microRNA-specific stem-loop structure primer. RESULTS: The PMCs changed to fusiformis following a high-glucose medium stimulated for 48 hours, and the EMT marker genes changed significantly, such as the decrease of E-cadherin and an increase of Vimentin (p < 0.01). These results proved the EMT had been induced by high-glucose. Applying real-time PCR with microRNA-specific stem-loop structure primer, miR-193a increased notably (p < 0.01), and miR-15a and let-7e decreased (p < 0.01), while miR-16 and miR-21 had no significant changes (p > 0.05). Most importantly, the increase of miR-193a was correlated with stimulus duration. CONCLUSIONS: MicroRNA with abnormal expression levels have a primary role in regulating the EMT of PMCs induced by high glucose.
OBJECTIVE: To determine the expression level of the microRNA in the process of epithelial-mesenchymal transition (EMT) of the peritoneal mesothelial cells (PMCs) induced by high glucose. MATERIALS AND METHODS: The PMCs were cultured using M199 medium with 10% fetal bovine serum, and the EMT was induced by D-glucose stimulation. Epithelial-mesenchymal transition was determined by changes in cell morphology and the expression levels of the EMT marker genes. Changes in cell morphology were observed by inverted microscope, and the expression levels of the EMT marker genes were determined by real-time PCR. The expression levels of the microRNA were detected by real-time PCR with microRNA-specific stem-loop structure primer. RESULTS: The PMCs changed to fusiformis following a high-glucose medium stimulated for 48 hours, and the EMT marker genes changed significantly, such as the decrease of E-cadherin and an increase of Vimentin (p < 0.01). These results proved the EMT had been induced by high-glucose. Applying real-time PCR with microRNA-specific stem-loop structure primer, miR-193a increased notably (p < 0.01), and miR-15a and let-7e decreased (p < 0.01), while miR-16 and miR-21 had no significant changes (p > 0.05). Most importantly, the increase of miR-193a was correlated with stimulus duration. CONCLUSIONS: MicroRNA with abnormal expression levels have a primary role in regulating the EMT of PMCs induced by high glucose.