MiR-148b Regulates Mitochondrial Fission In High Glucose Induced Human Renal Tubular Epithelial Cell Injury

Objective: To investigate the expression of microRNA-148b(miRNA-148b)in human renal tubular epithelial cells cultured in high glucose.To investigate the effects of microRNA-148 b on the injury of HK-2 cells and to explore the pathogenesis of tubular injury induced by high glucose and find new therapeutic targets.Methods: HK-2 cells were cultured in vitro and divided into the following groups after cell synchronization:(1)Normal glucose group(5.5 mmol/L glucose)and high glucose group(30.0 mmo/L glucose).After 48 hours of culture,the expression of microRNA-148 b was detected by RT-PCR.(2)Transfection of microRNA-148 b inhibitor inhibits the expression of microRNA-148 b.The cells were divided into normal glucose group,high glucose group,negative control high glucose group(transfection negative control + 30.0 mmo/L glucose),and microRNA-148 b inhibitor transfection high glucose group(transfection microRNA-148 b inhibitor + 30.0 mmol/L glucose)and cultured for 48 hours.MitoTracker fluorescent dye was used to detect the mitochondrial morphological changes under confocal microscopy;DCFH-DA fluorescent probe was used to detect the production of reactive oxygen species in each group under fluorescence microscopy.Western blot was used to detect the expression of AMPK alpha protein,DRP1 protein,Cytochrome C protein and cleaved-caspase3 protein,and Annexin v/FITC fluorescent dye was used to detect the apoptosis of cells in each group by flow cytometry.Results:(1)The expression of microRNA-148 b in high-glucose group was significantly higher than that in normal glucose group(p < 0.01).(2)The expression of AMPKα protein was down-regulated in high glucose group compared with normal glucose group.After inhibiting the expression of microRNA-148 b in high glucose group,the expression of AMPKα was up-regulated(p< 0.05),but still lower than that in normal glucose group.(3)The expression of mitochondrial fission related protein DRP1 increased in the high-glucose group,(p < 0.05).Compared with the high glucose group it decreased after inhibiting the expression of microRNA-148 b..(4)The mitochondria in the high glucose group were over-dividing,short rod or globular compared with the normal glucose group.After inhibiting the expression of microRNA-148 b in high glucose group,the over-division of mitochondria was inhibited,and more mitochondria were restored to linear and reticular.(5)ROS production increased and cytochrome C released more into cytoplasm in high glucose group compared with normal glucose group.After inhibiting the expression of microRNA-148 b in high glucose group,ROS production decreased and the release of cytochrome C protein to cytoplasm decreased.(6)Compared with the normal glucose group,the expression of cleaved-caspase 3protein and the apoptotic rate increased(p < 0.05).Compared with the normal glucose group,the expression of cleaved-caspase3 protein and the apoptotic rate decreased in the high glucose group(p < 0.05)after inhibiting the expression of microRNA-148 b in high glucose group.Conclusion:(1)The expression of microRNA-148 b in human epithelial cells cultured in vitro under high glucose was significantly up-regulated compared with that in normal glucose group.(2)Compared with the normal glucose group,the expression of AMPKα,target protein of microRNA-148 b,decreased,mitochondrial division increased,ROS production increased,cleaved-caspase3 protein expression increased,and apoptotic rate increased in vitro cultured human renal tubular epithelial cells under high glucose condition.Inhibiting the expression of microNA-148 b in human renal tubular epithelial cells under high glucose condition increased the expression of target protein AMPKαand inhibited mitochondrial division,reduced ROS production improved cell apoptosis and protected HK-2 cells from damage under high glucose conditions.MicroRNA-148 b may be a new therapeutic target for diabetic nephropathy..