Figure 6.

MAP4K4 influences MDM2 expression through regulating transcription factor c-Jun. (A) Gene expression profile array analysis in SW480 cells. Hierarchical clustering of the expression values for mRNA of siRNA-MAP4K4 cells vs. siRNA-NC cells. (B) Western blot analysis showed expression of MDM2, FAM172A, CAMLG, PLOD2 and MPEG1 protein after knockdown of MAP4K4. Silencing of transcription factor c-Jun resulted in downregulation of MAP4K4 expression in RKO and SW480 cells. (D) The human MDM2 promoter constructs containing a potential c-Jun binding motif (–1063 to –1057 bp). (E) The transcriptional activity of the MDM2 promoter. SW480 cells were co-transfected with the wild type or mutant type plasmids and c-Jun vector or blank vector for 48 h, and the luciferase activity was measured. (F) ChIP assays of c-Jun and its binding motif. Two antibodies (anti-IgG and c-Jun) were used in the ChIP assays in SW480 cells. QRT-PCR was performed to quantify the binding activity. (G) Products of qRT-PCR observed by electrophoresis method. (H) MAP4K4 knockdown influenced the expression of phosphorylated JNK and c-Jun, but had little effect on total JNK. MAP4K4-siRNA or siRNA-NC expressing SW480 transfectants were subjected to Western blot analysis. (I) Upregulation of miR-194 in SW480 cells had similar effects on expression of JNK, p-JNK and c-Jun to that of MAP4K4 knockdown. (J) The effects of overexpression of miR-194 on MDM2 expression. Representative images of 3 independent experiments with similar results are shown. **P<0.01.