Effects of miR-4429/NQO1 on high glucose-induced renal tubular epithelial injury and inflammatory factor secretion

Objective To investigate the effect of miR-4429/NQO1 and dependent reduced coenzyme/NADH quinone dehydrogenase 1 (NQO1) on the proliferation,apoptosis and inflammatory factor secretion of renal tubular epithelial cells induced by high glucose,and its mechanism.Methods Human renal tubular epithelial HK-2 cells were cultured in vitro and treated with 30 mmol/L glucose for 24 h to prepare a renal tubular epithelial cell injury model of diabetic nephropathy.The model rats were recorded as the high-glucose group.HK-2 cells were cultured for 24 h with the medium containing 5.5 mmol/L glucose and recorded as the normal control group.HK-2 cells were cultured with the medium containing 50 mmol/L mannitol for 24 h and recorded as the high-osmosis control group.The miR-4429 inhibitor (anti-miR-4429),negative control sequence (anti-miR-NC),small interfering RNA of anti-miR-4429 and NQO1(si-NOQ1),and negative control sequence of anti-miR-4429 and si-NQO1 (si-NC) were transfected into HK-2 cells and treated with 30 mmol/L glucose for 24 h.The rats treated as described above were recorded as HG + anti-miR-4429 group,HG + anti-miR-NC group,HG + anti-miR-4429 + si-NQO1 group and HG + anti-miR-4429 + si-NC group,respectively.qRT-PCR was used to detect the expression levels of miR-4429 and NQO1 mRNAs.Cell survival rate,apoptosis rate,Caspase-3 protein level,NQO 1 protein level,malondialedhyde (MDA) content,superoxide dismutase (SOD) activity,interleukin-1β (IL-1β) content,tumor necrosis factor-α (TNF-α) content were detected.The double luciferase reporting experiment validated the effect of miR-4429 overexpression on wild and mutant NQO1 luciferase activity.Western blot was used to detect the expression of Caspase-3 and NQO1.Results After high glucose treatment,the expression levels of miR-4429 and Caspase-3 were significantly increased (P<0.05),and the apoptosis rate was significantly increased (P<0.05).The content of MDA and the levels of IL-1β and TNF-α were significantly increased (P<0.05).but the expression level of NQO1 mRNA was significantly reduced (P<0.05),and the cell survival rate and SOD activity were significantly reduced (P<0.05).After inhibiting the expression of miR-4429,the apoptosis rate and the expression of Caspase-3 were significantly reduced (P<0.05),the content of MDA and the levels of IL-1β and TNF-α were significantly reduced (P<0.05),and the cell survival rate was significant It increased (P<0.05),and the activity of SOD increased significantly (P<0.05).Double luciferase reporting experiments confirmed that miR-4429 could inhibit luciferase activity in the 3'-UTR region of NQO1 (P<0.05).Inhibition of NQO1 expression could significantly reduce the protective effect of inhibition of miR-4429 expression on high glucose-induced renal tubular epithelial cell injury.Conclusions MiR-4429 can promote high glucose-induced apoptosis of renal tubular epithelial cells,reduce cell survival,and aggravate oxidative and inflammatory damage of cells through targeted regulation of expression of NQO1.