Effects of microRNA-133b on retinal vascular endothelial cell proliferation and apoptosis through angiotensinogen-mediated angiotensin II- extracellular signal-regulated kinase 1/2 signalling pathway in rats with diabetic retinopathy

Abstract

Purpose: This study aimed to explore the effects of microRNA-133b (miR-133b) on diabetic retinopathy (DR) by targeting angiotensinogen (AGT) through the angiotensin-II (AngII) extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway in rats.

Methods: The DR rat model was established using retinal tissues of DR rats and normal rats. Immunohistochemistry was performed to detect the protein expressions of AGT and CD34 in retinal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect miR-133b expression, AGT, AngII, ERK1/2 mRNA, and protein expressions in tissues and cells after transfection. Retinal vascular endothelial cells were cultured and divided into normal, blank, miR-133b mimics, miR-133b mimics negative control (NC), miR-133b inhibitors, miR-133b inhibitors NC, siRNA NC, siRNA-AGT, and miR-133b inhibitors + siRNA-AGT groups. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to detect cell proliferation. Cell cycle and apoptosis were evaluated using flow cytometry.

Results: Compared with normal rats, AGT and CD34 were expressed more frequently in DR rats. MicroRNA (miR)-133b expression was downregulated but AGT, AngII, ERK1 and ERK2 mRNA expressions were upregulated in retinal tissues of DR rats. When compared to the normal group, all other groups displayed decreased cell proliferation, increased cell number in G0/G1, decreased cell number in S stage, increased cell apoptosis rate and declined miR-133b expression. As well, significant increased expressions of AGT and the AngII-ERK1/2 pathway-related proteins were observed in retinal vascular endothelial cells in all groups except the normal group. The miR-133b mimics and siRNA-AGT groups had increased cell proliferation, decreased cell number in the G0/G1 phase, increased cell number in S stage, decreased cell apoptosis rate and decreased expressions of AGT and AngII-ERK1/2 pathway-related proteins than the miR-133b inhibitors + siRNA-AGT group. The miR-133b inhibitors group exhibited opposite trends compared with the miR-133b mimics and siRNA-AGT groups.

Conclusion: The study provides data to suggest that miR-133b induces proliferation and inhibits apoptosis of retinal vascular endothelial cells by targeting AGT through the AngII-ERK1/2 signalling pathway in DR rats.

Keywords: AngII-ERK1/2 signalling pathway; angiotensinogen; apoptosis; diabetic retinopathy; microRNA-133b; proliferation.