Figure 4
miR-1 silencing contributes to HIF-1α expression and phosphorylation of Smad3. (a) Predicted base pairing between miR-1 and 3′-UTR of HIF-1α. Wild-type or mutated (mut) 3′-UTR of HIF-1α sequence inserted into dual-luciferase vector were shown. (b) miR-1 mimics were co-transfected with psiCHECK2- HIF-1α-3′-UTR or psiCHECK2-HIF-1α-3′-UTR-mut, including mut1 and mut2, into HEK293 cells followed by dual-luciferase analysis. Data were presented as mean (±s.d.). n=3 in each group. ***P<0.001 as compared with indicated group. (Student’s t-test). (c,d) WB results show that compared with those treated with miR-1 mimics or miR-1 inhibitor transfection leads to changes in the protein levels of p-Smad3 in both HT-29 and CaCO2 cells. (e) Indicated group of HT-29 cells were extracted to detect nuclear and cytosolic Smad3 by western blotting, beta-actin and H3 were used as internal controls for cytosolic and nuclear fractions, respectively. (f) Immunofluorescence of Smad3 and HIF-1α localization in HT-29 and HCT-116 cell. (g) Smad3 interacted with HIF-1α. CaCO2 (upper) and HT-29 (bottom) cells were grown to 70–80% confluence, and then whole cell lysates were immunoprecipitated with anti-Smad3 antibodies and coprecipitated with HIF-1α. (h) The lysis of indicated group were immunoprecipitated with antibody targeting endogenous Smad3 and coprecipitated with HIF-1α by immunoblotting
