miR-15b/16 protects primary human retinal microvascular endothelial cells against hyperglycemia-induced increases in tumor necrosis factor alpha and suppressor of cytokine signaling 3

Abstract

Purpose: Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. Emerging evidence shows the impact of microRNA (miR) as a potential novel therapeutic target. The purpose of our study was to test the hypothesis that miR-15b and -16 are altered by hyperglycemia in retinal endothelial cells (REC), and that miR-15b/16 play key roles in in regulating insulin signaling through a reduction in TNFa- and SOCS3-mediated insulin resistance pathways.

Methods: Human REC were maintained in normal (5mM) glucose or transferred to high glucose medium (25 mM) for 3 days. REC were transfected with miRNA mimics (hsa-miR-15b-5p and hsa-miR-16-5p) 48 hours before cell harvest. A final concentration of 30 nM was used when transfected separately (miR-15b and -16) and 15 nM was used in combination (miR-15b + miR-16). A negative control group was treated with an equal concentration of a Mimic Negative Control. The levels of miRNA overexpression were verified using quantitative reverse transcription-polymerase chain reaction and real-time PCR. Western blot analyses were performed to study the levels of phosphorylated Akt (Serine 473), Akt, SOCS3, insulin receptor, phosphorylated insulin receptor (tyrosine 1150/1151), insulin receptor phosphorylated on Tyr960. In addition, ELISA was used to examine cleaved caspase 3 and TNFa. Analyses were done using unpaired Student t test. Data are presented as mean±SEM.

Results: We demonstrated that the expression of miR-15b and -16 was reduced in human REC cultured in hyperglycemia. Overexpression of miR-15b and/or -16 reduced TNFa and SOCS3 levels, while increasing IGFBP-3 levels and the phosphorylation of IR^Tyr1150/1151 in REC cultured in hyperglycemia. These, in turn, led to an increase of Akt phosphorylation and decreased cleavage of caspase 3

Conclusions: miR-15b and -16 play a role in the inhibition of insulin resistance via reduced TNFa- and SOCS3 signaling and increased IGFBP-3 levels, resulting in REC protection from hyperglycemia-induced apoptosis. This outcome suggests that both miR-15b and -16 are potential therapeutic targets for therapeutics for the diabetic retina.