Overexpression of Cullin7 is associated with hepatocellular carcinoma progression and pathogenesis

Abstract

Background: Overexpression of Cullin7 is associated with some types of malignancies. However, the part of Cullin7 in hepatocellular carcinoma remains unclear. The aim of this study was to investigate the role of Cullin7 in pathogenesis and the progression of hepatocellular carcinoma.

Methods: In the present study, the expression of Cullin7 in hepatocellular carcinoma cell lines and five surgical hepatocellular carcinoma specimens was detected with quantitative reverse transcription PCR and western blotting. In addition, the protein expression of Cullin7 was examined in 162 cases of archived hepatocellular carcinoma using immunohistochemistry.

Results: We found elevated expression of both mRNA and protein levels of Cullin7 in hepatocellular carcinoma cell lines, and Cullin7 protein was significantly upregulated in hepatocellular carcinoma compared with paired normal hepatic tissues. The immunohistochemistry analysis revealed that overexpression of Cullin7 occurred in 69.1% of hepatocellular carcinoma samples, which was a significantly higher rate than that in adjacent normal hepatic tissue (P < 0.01). Statistical analysis found that overexpression of Cullin7 was significantly associated with lymph node metastasis, tumor thrombus of the portal vein and advanced clinical stage (P < 0.05). Furthermore, by overexpressing Cullin7 in hepatocellular carcinoma HepG2 cells, we revealed that Cullin7 could significantly enhance cell proliferation, growth, migration and invasion. Conversely, knocking down Cullin7 expression with short hairpin RNAi in hepatocellular carcinoma HepG2 cells inhibited cell proliferation, growth, migration and invasion.

Conclusion: Our studies provide evidence that overexpression of Cullin7 plays an important role in the pathogenesis and progression of hepatocellular carcinoma and may be a valuable marker for hepatocellular carcinoma management.

Keywords: Cullin7; Hepatocellular carcinoma; Invasion; Proliferation; Western blot.

Fig. 1

Expression of Cullin7 is elevated in HCC. a Expression of Cullin7 protein in the normal human hepatic cell line LO2 and in the HCC cell lines HepG2, Hep-3B, HuH71 and SMMC-7721. The expression levels were normalized to β-actin. b Quantification of Cullin7 mRNA in LO2 and HCC cell lines. The expression levels were normalized to β-actin. Error bars represent standard deviations calculated from three parallel experiments. c The expression of Cullin7 protein in each of the HCC (T) and adjacent normal hepatic tissues (ANT) determined by western blotting. d Real-time PCR analysis of Cullin7 expression in each of the primary HCC (T) and paired hepatic adjacent non-cancerous tissues (ANT) from the same patient. e Quantification of Cullin7 protein in each of the primary HCC (T) and adjacent normal hepatic tissues (ANT) determined by western blotting. The expression levels were normalized to β-actin

Fig. 2

Positive expression of Cullin7 protein in HCC. a HE staining. The nuclei of tumor cells are large and have atypia, in contrast with those of adjacent normal hepatic tissues (HE 20 × 10). b Cullin7 immunohistochemical staining. Cullin7 expression is predominantly observed in the nuclei and is visualized as brown-yellow staining in HCC tissue. The expression of Cullin7 is negative in adjacent normal hepatic tissue (IHC 20 × 10)

Fig. 3

Upregulation of Cullin7 enhances the proliferation, migration, and invasion capacities of HCC cells. a Ectopic expression of Cullin7 in HepG2 cells analyzed by western blotting. β-actin was used as a loading control. b The transfection efficiency of Cullin7 was analyzed by measuring transcript levels using qRT-PCR analyses in HepG2 cells. c Cell proliferation after Cullin7 overexpression in cells was measured using MTT assays. d Colony formation assays show that upregulation of Cullin7 promotes cell growth, and the summary graphs are presented for the colony formation assay that is outlined. e HepG2 Cullin7 and control vector cells were subjected to a wound healing assay. f Overexpression of Cullin7 promoted cell invasion and migration as determined by Transwell migration and Matrigel invasion assays. Quantification of migrated cells through the membrane and invaded cells through Matrigel of each cell line are shown as a proportion of the vector controls. Error bars represent the mean ± SD of three independent experiments

Fig. 4

Knockdown of Cullin7 inhibited the proliferation, migration, and invasion capacities of HCC cells. a Knockdown of Cullin7 in specific shRNA-transduced stable HepG2 cells. β-actin was used as a loading control. b The transfection efficiency of shCullin7 was analyzed by measuring transcript levels using qRT-PCR analyses in HepG2 cells. c Silencing endogenous Cullin7 inhibited cell growth as determined using MTT assays. d Silencing endogenous Cullin7 inhibited cell growth as determined using colony formation assays. Summary graphs are presented for the colony formation assay that is outlined. e HepG2 shCullin7 and control vector cells were subjected to a wound healing assay (left panels). The uncovered areas in the wound healing assays were quantified as a percentage of the original wound area. f HepG2 shCullin7 and control vector cells were subjected to Transwell migration (upper panels) and Matrigel invasion assays (lower panels). Quantification of migrated cells through the membrane and invaded cells through Matrigel of each cell line are shown as a proportion of the vector controls. Error bars represent the mean ± SD of three independent experiments