Figure 4.

miR-122/SENP1 axis regulates the Wnt/β-catenin signaling pathway through the de-SUMOylation effect of SENP1 on β-catenin. (A) Western blotting was performed to measure the Wnt1 and β-catenin protein expressions in HepG2 cells transfected with the miR-122 mimic and/or SENP1 overexpression vector. Untransfected cells were considered as the control. (B) HepG2 cell lysates were immunoprecipitated with control IgG, anti-SENP1 and anti-β-catenin antibodies. The immunoprecipitates were subsequently immunoblotted with anti-SENP1 and anti-β-catenin antibodies. (C) FLAG-tagged SENP1 and/or Myc-tagged β-catenin were transfected into HepG2 cells before being lysed and immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were subsequently immunoblotted with anti-SENP1 and anti-Myc antibodies. The whole-cell lysate of HepG2 cells served as input. The lysate from HepG2 cells transfected with FLAG-tagged SENP1 or Myc-tagged β-catenin alone served as the negative control. (D) HepG2 cells were transfected with the indicated constructs and treated with MG132 for 6 h before in vitro SUMOylation assay. (E) HepG2 cells were transfected with either the empty or SENP1 overexpression vectors. After 48 h, cells were treated with CHX for 0, 2, 4 and 8 h, before being harvested, lysed and the proteins detected by western blot analysis for β-catenin. *P<0.05 and ***P<0.001 vs. the control; ^###P<0.001 vs. the miR-122. miR, microRNA; SENP1, sentrin-specific protease 1; IgG, immunoglobulin G; CHX, cycloheximide.