Effects of miR-141 silencing on renal tubular epithelial cell damage induced by high glucose and Sirt1/Nrf2 signaling pathway / 中华内分泌外科杂志

ABSTRACT

Objective:To investigate the effect of miR-141 down-regulation on the damage of renal tubular epithelial cell, and further to explore its mechanism.Methods:The renal tubular epithelial cell line HK-2 cells were divided into normal (5.5 mmol/L D-glucose) group, hypertonic group, high glucose (30 mmol/L D-glucose) group, negative control+high glucose group (transfected with NC inhibitor vector) and si-miR-141+high glucose group (transfected with miR-141 inhibitor vector) . Real time quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression of miR-141. The production of reactive oxygen species (ROS) , cell viability and apoptosis were detected by DCFH-DA fluorescence staining, CCK-8 method and flow cytometry. The expression of Sirt1/Nrf2 signaling pathway related proteins was detected by Western blot. Luciferase reporter gene assay verified the targeting relationship between miR-141 and Sirt1 mRNA.Results:①Compared with the normal group, after transfection with si-miR-141, the relative expression of miR-141 decreased (1.00±0.03 vs 0.52±0.06) , the difference was statistically significant ( F=278.104, P<0.05) ; ② Compared with the normal group [DCFH-DA fluorescence intensity (7.18±0.59) %], the high glucose group [DCFH-DA fluorescence intensity (84.95±3.21) %] cell ROS level was significantly increased, and compared with the high glucose group [DCFH-DA fluorescence intensity (84.95±3.21) %] Compared with the si-miR-141+ high glucose group [DCFH-DA fluorescence intensity (45.10±4.29) %] cell ROS levels were significantly reduced, the difference was statistically significant (all P<0.05) ; ③compared with the normal group (5.13%±0.78) % Compared with the hypertonic group (5.96±0.81) %, the high glucose group (32.76±2.95) % cell apoptosis rate was significantly increased, while the si-miR-141+ high glucose group (17.54%± 2.79) % apoptosis rate was significantly lower in the higher glucose group and the negative control+ high glucose group (33.40%±3.14) %, the difference was statistically significant ( F=221.419, P<0.05) ; ④compared with the normal group (100±3.98) % Compared with the hypertonic group (95.68±5.14) %, the high glucose group (67.24±5.18) % HK-2 cell survival rate was significantly reduced; at the same time, compared with the high glucose group (67.24±5.18) % and Compared with the negative control+ high glucose group (65.33±3.10) %, the si-miR-141+ high glucose group (83.55±5.10) % cell survival rate increased significantly, and the difference was statistically significant ( F=93.008, P<0.05) ; ⑤ Compared with the normal group and the hypertonic group, the expression of Cleaved Caspase 3 protein in the high glucose group increased, while the expression of Sirt1, Nrf2 and HO-1 protein was down-regulated; however, compared with the high glucose group, si- In the miR-141+ high glucose group, Cleaved caspase 3 protein expression decreased, while Sirt1, Nrf2 and HO-1 protein expression increased, the difference was statistically significant (all P<0.05) . Conclusions:Down-regulation of miR-141 can ameliorate high glucose-induced renal tubular epithelial cell damage induced oxidative stress by activating Sirt1/Nrf2 signaling pathway.