Fig. 5
KPC1 p.M8V editing weakens the p50-mediated inactivation of NF-κB signaling. a KPC1-associated biological processes discriminated by gene set enrichment analyses (GSEA). GSEA was performed on the basis of the mean of the KPC1 mRNA expression levels in iCCA tissues from the patients of DISC cohort (KPC1High versus KPC1Low). NES, normalized enrichment score. b GSEA plots of the signatures of the epithelial-mesenchymal transition (Left panel) and TNF-α signaling via NF-κB (Right panel). c and d Immunoblotting (C) and immunofluorescence (D) assays in QBC939 cells showed that knockdown of KPC1 induces epithelial-mesenchymal transition (EMT) evidenced by epithelial marker E-cadherin and mesenchymal marker N-cadherin or Vimentin. The arrows in (D) mark spindle-shaped cells. Scale bar in (D), 20 µm. e Luciferase reporter assays showing that knockdown of KPC1 enhances the NF-κB signaling in QBC939 cells. f Knockdown of KPC1 induces the mRNA expression levels of multiple downstream targets of p50, including IL6, IL6R, MMP9 and VEGFA. g Chromatin immunoprecipitation (ChIP)-qPCR assays in QBC939 cells show that KPC1 knockdown weakens the binding of p50 to the promoters of its targets IL6, IL6R, MMP9 and VEGFA. h Luciferase reporter assays in QBC939 cells show that KPC1 p.M8V editing (KPC1-EDT) abolishes the inhibitory effect on NF-κB signaling by wide-type KPC1 (KPC1-WT). i Real-time PCR assays in QBC939 cells show that KPC1-EDT abolishes the inhibitory effect on expression of multiple downstream targets of p50 by KPC1-WT. j ChIP-qPCR assays in QBC939 cells show that KPC1-EDT abolishes the promoting effect on binding of p50 to the promoters of its targets by KPC1-WT. Data are presented as the mean ± standard deviation (s.d.). *P < 0.05, **P < 0.01 and ***P < 0.001; assessed by Student’s t test