Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-κB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications

Abstract

The link of proto-oncogenic protein Wnt-1 production with NF-kappaB activation has been functionally demonstrated in PC12 cells, a rat pheochromocytoma cell line of neural crest lineage, while it is not yet verified in human cells. The link can be indirectly supported in our previous report that functional proteomics identifies enhanced expression of NF-kappaB-associated Wnt-1 production in human hepatocellular carcinoma tissues. This study aimed to further validate this link in human cells using anti-sense strategy. The effects of sequence-specific anti-sense morpholino oligonucleotides (ONs) targeting against pre-mRNA sequences of human p50 and p65 subunits of NF-kappaB as well as Wnt-1 genes were investigated. It revealed that all the three morpholino ONs inhibited NF-kappaB activation in human hepatoblastoma cell line HepG2 cells along with decreased Wnt-1 production. Chromatin immunoprecipitation assay ascertained the direct binding of NF-kappaB-p50 to the Wnt-1 promoter. Additionally, anti-P50 and anti-P65 morpholino ONs also repressed the phosphorylation of Ikappa Balpha which temporarily correlated with the inhibition of NF-kappaB activation accompanied by decreased Wnt-1 production by HepG2 cells. In summary, NF-kappaB activation is critically involved in the production of Wnt-1 by HepG2 cells. These results may have important oncology implications in treating patients with NF-kappaB-associated Wnt-1-producing cancers.

The vignette for V15 N5 issue

“…Anti-sense morpholino oligonucleotide assay shows critical involvement for NF-jB activation in the production of Wnt-1 protein by HepG2 cells: oncology implications The present study [21] is to validate the previous finding by functional proteomics that Wnt-1 enhances the expression of NF-jB in human hepatocellular carcinoma tissues. By using anti-sense morpholino oligonucleotides(ONs) targeting pre-mRNA sequences of human p50 and p65 subunits of NF-jB as well as Wnt-1 genes, it revealed that the three ONs inhibited NF-jB activation in HepG2 cells and decreased Wnt-1 production.…”

Wnt-1 Protein as a Prognostic Biomarker for Hepatitis B–Related and Hepatitis C–Related Hepatocellular Carcinoma after Surgery

“…Enhanced expression of nuclear factor-nBassociated Wnt-1 protein has been identified in a large proportion of HCC specimens with HBV and/or HCV infection (18). The nuclear factor-nB -P50 subunit has been shown to bind to the promoter of the Wnt-1 gene by chromatin immunoprecipitation assay (24). All evidence highly suggests that Wnt-1 plays an important role in HBV-and/or HCV-related hepatocarcinogenesis.…”

Wnt/β-catenin signaling as a useful therapeutic target in hepatoblastoma

“…An increase in activation of Wnt/β-catenin signaling may also be secondary to activation of GPC3, NF-kB, GSK-3β, ERK1/2 and co-activator of β-catenin, i.e. FHL2 [36,42,46]. The activation of β-catenin signaling may trigger the activation of other signaling pathways to induce the development of hepatoblastoma, including inhibition of TNF-α-dependent apoptosis, increase in the secretion of epithelial growth factor and formation of complex of with YAP-1 protein [40,47,51].…”

“…Chromatin immunoprecipitation assay revealed the direct binding of NF-kB-p50 to the Wnt-1 promoter region. Accordingly, it may be suggested that the activation of NF-kB may increase the production of Wnt-1, which triggers the activation of Wnt/β-catenin to induce the development of hepatoblastoma [46].…”

“…In hepatoblastoma cell lines (HuH-6 and HepG2) transfection of siRNAs or sequence-specific antisense morpholino oligonucleotides leads to degradation of overexpressed β-catenin in the nucleus and reduces the levels of c-myc. It suggests that β-catenin may be potential target of gene therapy to manage pediatric hepatic tumors, with mutations and overexpression of β-catenin gene [46,55]. Transfection of hepatoblastoma cell lines (Huh-6 and HepG2) with siRNA of frizzled receptors (receptor of Wnt ligands) is shown to inhibit Wnt signaling, decrease the expression of frizzled receptor genes, cyclin D1 and suppress the cellular proliferation.…”