Research Paper Advance Articles

Figure 4. NUMB is a direct miR-222 target in human retinal pigment epithelial cells. (A) A Venn diagram shows the numbers of candidate miR-222 mRNA targets predicted by three public algorithms: TargetScan, miRDB, and micro-T. (B) The mRNA and (C) protein levels of the indicated candidates in ARPE-19 cells were determined 48 h after transfection of control (NC) or miR-222 mimics. (D) Diagrams show the putative miR-222 binding sites and corresponding wild type (WT) or mutant (MUT) sites of NUMB 3’UTR. The luciferase activity was detected in ARPE-19 cells at 48 h after co-transfection of WT or MUT NUMB 3’UTR together with negative control (NC) or miR-222 mimics. (E) NUMB protein levels in ARPE-19 cells treated with palmitic acid at the indicated concentrations. (F) ARPE-19 cells were transfected with negative control or miR-222 mimics, and 48 h later cells were left untreated or treated with 200 μM palmitic acid for another 48 h. NUMB protein levels in each group were determined by western blot. For all western blot data, representative bands images are shown, and the relative protein levels were summarized from three independent experiments with similar results. n = 3 for each group; ^*P < 0.05, ^**P < 0.01. Abbreviation: ns: not significant, between the indicated groups.