Figure 3.

ROCK1 is a target gene of miR-506 in neuroblastoma cells. (A) According to bioinformatics analysis, the 3′-UTR of the ROCK1 gene contains binding sites for miR-506. (B) miR-506 suppressed the expression of a luciferase reporter gene harboring the 3′-UTR of ROCK1 in IMR-32 cells. The pGL4 plasmid was modified by adding either the human 3′-UTR or the 3′-UTR with mutations in regions complementary to miR-506 seed regions behind the firefly luciferase gene. IMR-32 cells were transiently co-transfected with a negative control (mock) or miR-506 together with the indicated luciferase constructs, and luciferase activity was analyzed 48 h later. Data are presented as relative firefly luciferase activity normalized to Renilla luciferase activity from the same construct. (C) miR-506 restoration downregulated ROCK1 in the neuroblastoma cells. The cells were transfected with miR-506 or miR control for 48 h, and were then collected for reverse transcription-quantitative polymerase chain reaction. (D) miR-506 restoration downregulated ROCK1 expression in the neuroblastoma IMR-32 and SH-SY5Y cell lines. The cells were transfected with miR-506 or miR-control for 48 h, and were then collected for western blot analysis. The data are presented as the mean ± standard deviation and the data were collected from three independent experiments. ROCK1, Rho-associated, coiled-coil containing protein kinase 1; 3′-UTR, 3′-untranslated region; miR, microRNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.