Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration

Abstract

Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.

Fig. 1

ZNF 267 expression in HCC. (A) Expression of ZNF267 mRNA in primary human hepatocytes (PHH) and four HCC cell lines (HepG2, Hep3B, PLC, HUH-7) analyzed by quantitative real-time PCR. (B) ZNF267 mRNA expression in human HCC tissue specimens and corresponding non-tumorous tissue samples of 11 patients. (C) Expression of ZNF267 mRNA in PHH and activated human hepatic stellate cells (HSC) of 2 different donors. (D) Correlation of ZNF267 and collagen I mRNA expression in cirrhotic liver tissue of 11 patients.

Fig. 2

Regulation of ZNF267 expression in HCC cells. (A) Analysis of ZNF267 mRNA expression in Hep3B cells transiently transfected with an Ets-1 expression plasmid. (*: p < 0.05 compared to control plasmid pcDNA). (B) ZNF267 promoter activity in Hep3B cells transiently transfected with luciferase reporter plasmids containing the ZNF267 promoter or the same construct with a mutation in the Ets-1 binding site. (*: p < 0.05). (C) ZNF267 mRNA expression in HCC cells with or without pharmacological induction of hypoxia by DP (2,2′-dipyridyl), or HIF1alpha inhibition by echinomycin (Ech). (*: p < 0.05). (D) Expression of ZNF267 mRNA in HCC cells at different time points after incubation with the ROS inducer arsenic acid. (*: p < 0.05 compared to control). (E) Analysis of ZNF267 mRNA expression in HCC cells after treatment with the ROS scavenger histidine. (*: p < 0.05 compared to 0 μM histidine).

Fig. 3

Functional effects of ZNF267 suppression in HCC cells. (A) ZNF267 mRNA expression in HCC cells transiently transfected with two different ZNF267 siRNAs (siRNA1 and siRNA2) and cells transfected with control siRNA. (B) Microscopical analysis of Hep3B cells after ZNF267 suppression. Analysis of snail-1 (C) and slug (D) mRNA expression in HCC cells transiently transfected with ZNF267 siRNA. (E) Proliferation of HCC cells after ZNF267 suppression determined by analysis of XTT activity. (F) Migratory activity of siRNA-treated HCC cells assessed with the Boyden chamber assays. (*: p < 0.05 compared to control).

Fig. 4

Effect of ZNF267 overexpression in HCC cells. (A) ZNF267 mRNA expression in Hep3B cells transiently transfected with a ZNF expression plasmid and cells transfected with the empty vector. (B) Microscopical analysis of Hep3B cells after overexpression of ZNF267. Analysis of snail-1 (C) and slug (D) expression in HCC cells transiently transfected with ZNF267 expression plasmid. (E) Proliferation of Hep3B cells overexpressing ZNF267 determined by analysis of XTT activity. (F) Migratory activity of Hep3B cells after ectopic expression of ZNF267 assessed with the Boyden chamber assays. (* p < 0.05 compared to control).