Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose

Abstract

AIM: To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. METHODS: hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. RESULTS: Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. CONCLUSION: Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.