Figure 4

FOXO1 is a direct target of miR-1269. A. Sequence alignment of miR-1269, miR-1269 mutant (miR-1269-mut), and putative FOXO1-3’UTR. B. Real-time PCR analysis of miR-1269 and western blotting analysis of FOXO1 in 8 paired cancerous tissues (T) and their adjacent noncancerous hepatic tissues (N). The quantification of western blotting bands is performed by OD value determined by Quantity One 4.6.2 software. C. Western blotting analysis of expression levels of FOXO1 and phosphorylated FOXO1 in the indicated cells. α-Tubulin served as the loading control. D. The FOXO1 luciferase reporter activity in the indicated cells transfected with miR-1269 mimic, or miR-1269-mut, miR-1269 inhibitor, or negative control. E. Real-time PCR analysis of mRNA expression of p21, Cyclin D1 and Rb in the indicated HCC cell lines. F. Western blotting analysis of p21, cyclin D1, Ki67, p-Rb, and Rb in the indicated HCC cells. α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.