OncomiR Addiction Is Generated by a miR-155 Feedback Loop in Theileria-Transformed Leukocytes
Figure 5
miR-155 stabilized c-Jun by inhibiting its proteasomal degradation.
(A) Overexpression of miR-155 or depletion of DET1 in BL3 cells increased the half-life of endogenous c-Jun protein. BL3 cells transiently expressing miR-155 or siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semi-quantification with an αTubulin antibody as a loading control. Relative c-Jun protein levels at time 0 were set as 1 (average ± sd, n = 3). (B) Inhibition by the miR-155 Sponge in TBL3 cells decreased the half-life of endogenous c-Jun. These effects were reversed by siRNA against DET1. TBL3 cells transiently expressing miR-155 Sponge +/− siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semiquantification with α-Tubulin as a loading control. Relative c-Jun levels at time 0 were set as 1 (average ± sd, n = 3). (C) Effect of the miR-155 Sponge on c-Jun protein levels was rescued by treating the proteasome inhibitor MG132. TBL3 cells transiently expressing the miR-155 Sponge were treated with MG132 for 3 h, followed by immunoblot analysis with the c-Jun antibody and semiquantification with α Tubulin as a loading control (average ± sd, n = 3). (D) Overexpression of miR-155 or depletion of DET1 in BL3 cells reduced c-Jun ubiquitination. Transfected cells were treated with MG132 for 3 h, followed by endogenous c-Jun immunoprecipitation and immunoblot analysis with indicated antibodies (average ± sd, n = 3). *p<0.05, **p<0.01, ***p<0.001.