Fig. 6

RACK1 and miR-124 are required for the suppressive effects of DKC1125 on cell invasiveness. a RACK1 in the KITENIN complex plays a major role in the inhibition of cell invasion by DKC1125. Cell invasion was examined in empty vector (EV)- or KITENIN-transfected Caco2 cells after knockdown of RACK1 via siRNA transfection (left panel), or under ectopic expression of RACK1 (right panel) after treatment with vehicle (V) or DKC1125 (D) (0.5 μM). Data are expressed as in Fig. 1a. b Dvl2 downregulation by DKC1125 was associated with elevated binding of Dvl2 to RACK1. Several deletion mutants within the binding site of KSRP to DKC1125 were designed and co-expressed in Caco2 cells with RACK1-GFP. Dvl2–RACK1 binding was examined using GFP-Trap and immunoblot analysis after treatment with DKC1125 (0.5 μM), and compared with that of empty vector (EV) or wild-type (WT) KSRP expression. An increase in Dvl2–RACK1 interaction was observed after DKC1125 treatment in cells expressing the Q417A-KSRP or N467A-KSRP mutant, but not in cells expressing the R411A-KSRP or R415A-KSRP mutant. c Modulation of miR-124 is also involved in increased cellular invasiveness by the functional KITENIN complex. Detection of transcript of miR-124-3p in stably miR-null- or miR-124-transfected Caco2 cells (left panel). Cell invasion was examined in Caco2 cells stably expressing the miR-null vector or miR-124 that were transfected with the empty vector (EV)-, KITENIN-, WT-KSRP-, or Δ34KH-KSRP, and treated with vehicle or DKC1125 (0.5 μM) (right panel). Data are expressed as in Fig. 1a. d Inhibitor of miR-124-3p significantly restored the inhibitory effect of DKC1125 on the KITENIN-mediated increase in cell invasion. Cell invasion was compared in empty vector (EV)- or KITENIN-transfected Caco2 cells treated with vehicle or DKC1125, or co-treated with DKC1125 (0.5 μM) and a synthetic-oligo inhibitor of miR-124-3p (50 nM). The asterisk indicates a significant difference in Caco2/KITENIN-V5 cells after treatment with DKC1125, and a significant difference in DKC1125-treated Caco2/KITENIN-V5 cells after treatment with a synthetic-oligo inhibitor of miR-124-3p (**P < 0.01). Data are expressed as in Fig. 1a