Figure 3.

CDK2 and PAF are direct targets of miR-200b. (A) Putative miR-200b binding sites on the 3′UTRs of CDK2 and PAF mRNAs are shown, and the deletion mutation sites of the luciferase reporters are indicated by asterisks. (B) Thirty-six hours after transfection, dual luciferase reporter assay was performed to measure the impact of miR-200b on the 3′UTR of CDK2 and PAF in EC109 cells. WT, wild-type; Mut, mutation. (C–E) ESCC cells were transfected with miR-200b mimics or negative control RNA (30nM), 36h after transfection, the expression of protein and mRNA was determined by Western blot (C and D) and real-time PCR (E), respectively. In Western blot assays, Actin was used as a loading control for whole cell lysates, while α-Tubulin and HDAC1 were used as loading controls for cytoplasmic (Cyto) and nuclear lysates, respectively. GAPDH was used as a loading control for real-time PCR. Representative data are shown, and data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.