Figure 4

miR-208 directly targets the 3′-UTR of SOX6 mRNA. A. Schematic representation of the miR-208 target sites in the 3′-UTR of SOX6 mRNA and a miR-208 mutant containing two altered nucleotides in the seed sequence (miR-208-mut). B. The expression levels of SOX6 protein in Kyse30 and Kyse410 cells overexpressing or suppressing miR-208, by western blotting 48 hours after transfection. α-Tubulin served as the loading control. C. Luciferase assay of indicated cells transfected with the pGL3-SOX6-3′UTR reporter. D. Real-time PCR analysis of SOX6 downstream genes mRNA expression in indicated ESCC cells. E. Expression of p21, CyclinD1, phosphorylated pRb, and Rb protein as measured by western blotting in indicated ESCC cells. α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. *P <0.05.