Reduced IκBα promotes hepatocellular carcinoma cell proliferation and migration via regulation of NF-κB/Erbin axis

Abstract

Aberrantly low expression of NF-κB inhibitor α (IκBα) is observed in hepatocellular carcinoma (HCC), yet the underlying mechanism via which IκBα regulates HCC remains largely unknown. Therefore, to determine the potential function of IκBα in hepatocarcinogenesis, the present study used immunohistochemistry (IHC) staining to analyze the associations between IκBα protein expression and clinicopathologic characteristics of 107 patients with HCC. It was found that expression of IκBα was significantly associated with tumor recurrence. Moreover, IκBα protein expression was decreased in 107 HCC tissue samples and was positively associated with overall survival. Mechanistically, it was demonstrated that silencing of IκBα activated NF-κB in both Huh7 and HCCLM3 cells, followed by upregulation of Erbb2 interacting protein (Erbin) at both the mRNA and protein levels, confirmed by reverse transcription-quantitative PCR and western blotting, to promote cell proliferation and migration. Furthermore, knockdown of Erbin significantly attenuated NF-κB-mediated cell proliferation and migration. It was also identified that overexpression of Erbin in HCC tissues promoted both cell proliferation and migration, and was negatively associated with IκBα expression in 107 HCC tissue samples. Thus, these results indicated that downregulation of IκBα promoted HCC tumorigenesis via upregulation of NF-κB-mediated Erbin expression.

Keywords: Erbb2 interacting protein; HCC; IκBα; tumorigenesis.

Figure 1.

IκBα expression is downregulated in HCC and associated with poor prognosis of patients with HCC. (A) Representative images of HCC and adjacent liver tissues from 107 cases stained with anti-IκBα antibody. (B) Association between OS time of 107 patients with HCC and IκBα expression. Cells were transfected with (C) siIκBα or (D) HA-IκBα and proliferation was assessed after 24 h of transfection using Cell Counting Kit-8 assay. Cells were transfected with (E) siIκBα or (F) HA-IκBα for 48 h and proliferation was assessed with a clonogenic survival assay. Cells were transfected with (G) siIκBα or (H) HA-IκBα for 48 h and then analyzed with a Transwell migration assay. Scale bar, 100 µm. Error bars represent SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. corresponding control group. siRNA, small interfering RNA; IκBα, NF-κB inhibitor α; Cont, control; HCC, hepatocellular carcinoma; OS, overall survival; HA-IκBα, overexpression plasmid.

Figure 2.

IκBα negatively regulates Erbin expression via NF-κB. (A) Representative images of HCC and adjacent liver tissues from 107 cases stained with anti-Erbin antibody. (B) Association between OS time of 107 patients with HCC and Erbin expression. Cells were transfected with (C) siIκBα or (D) HA-IκBα for 48 h and Erbin expression was determined using western blot analysis. Cells were transfected with (E) siIκBα or (F) HA-IκBα for 48 h and NF-κB p65 expression was determined using western blot analysis. (G) Cells were transfected with siIκBα for 48 h and mRNA expression levels of c-Rel and Ezh2 were assessed using RT-qPCR. Cells were transfected with (H) Flag-NF-κB p65 or (I) siNF-κB p65 for 48 h and then the mRNA expression of Erbin was measured using RT-qPCR. Cells were transfected with (J) Flag-NF-κB p65 or (K) siNF-κB p65 for 48 h, then Erbin expression was determined using western blot analysis. Scale bar, 100 µm. The error bars represent SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. corresponding control group. siRNA, small interfering RNA; IκBα, NF-κB inhibitor α; Cont, control; HCC, hepatocellular carcinoma; OS, overall survival; Erbin, Erbb2 interacting protein; RT-qPCR, reverse transcription-quantitative PCR; Rel, Rel proto-oncogene, NF-kB subunit; Ezh2, enhancer of zeste 2 polycomb repressive complex 2 subunit.

Figure 3.

IκBα is negatively associated with Erbin in patients with HCC. (A) Representative images of HCC and adjacent liver tissues from 107 cases stained with anti-IκBα and anti-Erbin antibodies. (B) There was a negative association between IκBα and Erbin expression levels. Scale bar, 100 µm. Erbin, Erbb2 interacting protein; IκBα, NF-κB inhibitor α; HCC, hepatocellular carcinoma.

Figure 4.

Erbin is responsible for NF-κB-mediated HCC cell proliferation and migration. Cells were transfected with (A) HA-Erbin or (B) siErbin for 48 h and Erbin expression was determined by western blotting. Cells were transfected with (C) HA-Erbin or (D) siErbin and cell proliferation was assessed after 24 h of transfection using CCK-8 assay. After transfection with (E) HA-Erbin or (F) siErbin for 48 h, cells were seeded into Transwell plate for 24 h and stained with 0.1% crystal violet staining solution. Cells were transfected with Flag-NF-κB p65, with or without Erbin siRNA, and analyzed using (G) western blotting, (H) CCK-8 assay or (I) Transwell migration assay. Scale bar, 100 µm. The error bars represent SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. corresponding control group. siRNA, small interfering RNA; IκBα, NF-κB inhibitor α; Cont, control; Erbin, Erbb2 interacting protein; CCK-8, Cell Counting Kit-8.