Figure 4

Luciferase assay demonstrates that miR-1 directly targets PTMA 3' UTR. (A) Schematic representation of the miR-1 targeting sequences that complement with the 3' UTR of PTMA mRNA. The PTMA 3'UTR (+779~801 and +973 ~ +997 nt, miR-1 predictive binding site) were cloned into pmirGLO luciferase vector to generate pmir-PTMA#1-wt and pmir-PTMA#2-wt. The miR-1 seed region complementary sequences were mutated and named as pmir-PTMA#1-mut and pmir-PTMA#2-mut. (B) NPC-TW01 cells were transfected with miR-1 or without any microRNA. NPC-TW01 cells were then transfected with pmirGLO empty vector, pmir-PTMA#1-wt, pmir-PTMA#2-wt, pmir-PTMA#1-mut or pmir-PTMA#2-mut. Luciferase assays were performed after 36 hours of transfection. The results indicate that miR-1 directly binds to the PTMA mRNA 3'UTR and impairs PTMA expression in NPC-TW01 cells. (*Comparison between two groups as indicated, p < 0.05. Error bars correspond to mean ± SD)