Negative regulation of microRNA-709 in mitochondrial function participating in the renal tubular epithelial cell injury

Abstract

Objective To investigate the role of microRNA-709 (miR-709)in murine renal tubular epithelial cells with Cisplatin treatment and its underlying mechanism. Methods The cells were cultured with Cisplatin in the concentrations (0, 1, 5, 10, 20 μmol/L) for 24 h in vitro, and underwent 10 μmol/L Cisplatin stimulation at diffe-rent time points (0, 2, 6, 12, 24 h). Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to investigate the level of miR-709.mPTC were divided into a negative control group, miR-709 mimic group, inhibitor negative control (INNC)group, miR-709 inhibitor group, INNC group and miR-709 inhibitor group were treated with Cisplatin treatment.Annexin V-FITC/PI double staining was applied to detect apoptosis.Caspase-3 activity was detected by spectrophotometry.The mRNA and protein levels of E-cadherin were detected by RT-PCR and Western blot.Mitochondrial membrane potential and mitochondrial superoxide (mitoSOX) generation were detected by flow cytometry.Mitochondrial DNA(mtDNA) copy number was verified by PCR. Results The level of miR-709 increased in Cisplatin 5 μmol/L stimulation group(F=22.17, P<0.05)and increased further 12 h later after cultured in 10 μmol/L Cisplatin compared with that of the control group(F=33.462, P<0.05). After overexpression of miR-709, apoptotic rate and Caspase-3 activity of the mPTC increased, while the expression of E-cadherin declined when compared with that in the negative control group (apoptotic rate: 1.54±0.20 vs 1.00±0.23; Caspase-3 activity: 1.27±0.08 vs 0.97±0.08; E-cadherin: 0.47±0.15 vs 1.00±0.10; t=-3.086, -5.882, 5.671, all P<0.05). In addition, the levels of JC-1 and mtDNA decreased while mitoSOX increased compared with that of the control group (JC-1: 0.80±0.04 vs 1.05±0.08; mtDNA: 0.58±0.15 vs 1.00±0.75; mitoSOX: 1.31±0.16 vs 1.00±0.05; t=4.687, 4.943, -3.694, all P<0.05). Downregulation of miR-709 attenuated the tubular cell injury and the mitochondrial dysfunction was induced by Cisplatin (apoptotic rate: 1.35±0.10 vs 1.86±0.44; Caspase-3 activity: 1.04±0.12 vs 1.30±0.09; E-cadherin: 0.86±0.08 vs 0.54±0.05; JC-1: 0.94±0.06 vs 0.75±0.05; mtDNA: 0.68±0.09 vs 0.27±0.12; mitoSOX: 0.91±0.09 vs 1.22±0.08; F=18.489, 20.932, 33.323, 21.726, 59.330, 23.813, all P<0.05). Conclusion Mitochondrial function negatively regulated by miR-709 may be involved in the renal tubular epithelial cell injury induced by Cisplatin. Key words: Cisplatin; Mitochondrial function; MicroRNA-709; Renal tubular epithelial cell injury