Fig. 2.

MiR-15a negatively regulates the expression of ASM by direct targeting 3′UTR of ASM mRNA

(A) Expression profiles of miR-15a and ASM were determined by real-time PCR in HRECs from control donors (n = 6) and HRPE cells (n = 3). (B) ASM expression was examined in HRECs from diabetic donors (n = 6) compared with control donors (n = 6). (C) Expression profiles of miR-15a and ASM were detected by real-time PCR in HRPE treated with 25 mmol/l glucose for 24 h compared with 5 mmol/l glucose (n = 3). (D) HRPE cells were co-transfected with wild type human ASM 3′UTR or mutant ASM 3′UTR (200 ng) luciferase reporters and miR-15a mimic in increasing concentrations (30, 50 and 100 nM). Mutant human ASM 3′UTR and control mimic served as negative controls. Luciferase activity was measured 48 h post-transfection. (E) Real-time PCR analysis of ASM mRNA level after miR-15a mimic or inhibitor delivery in HRECs. (F) Real-time PCR analysis of ASM mRNA level after miR-15a mimic or inhibitor delivery in HRPE cells under normal and high glucose conditions for 24 h (n = 3). (G) Western blot analysis of ASM protein expression after miR-15a mimic or inhibitor delivery in HRPE cells under normal and high glucose conditions for 48 h (n = 3). α-tubulin serves as a loading control. Representative blots are from three independent experiments. Quantification of band intensity is relative to control. Data are mean ± SEM (n = 3). *** P < 0.001; ** P < 0.01; * P < 0.05; not significant at P > 0.05.