ZNF521 which is downregulated by miR-802 suppresses malignant progression of Hepatocellular Carcinoma through regulating Runx2 expression

Abstract

Zinc finger protein 521 (ZNF521) plays an important role in the tumor development and process. However, its regulatory role in hepatocellular carcinoma (HCC) remains unclear. In this study, we demonstrated for the first time that ZNF521 mRNA and protein was down-regulated in HCC tissues and cell lines. Down-regulated ZNF521 expression was significantly associated with malignant prognostic features, including advanced TNM stage and large tumor size. For 5-year survival, ZNF521 served as a potential prognostic marker of HCC patients. Moreover, ZNF521 inhibited cell proliferation, colony formation and cell viability through Runx2 transcriptional inhibition and AKT phosphorylation pathway. Moreover, we demonstrated that ZNF521 expression was regulated by miR-802. In HCC tissues. MiR-802 has an inverse correlation with ZNF521 expression. In conclusion, we demonstrate for the first time that ZNF521 is down-regulated in HCC tissues and inhibits HCC growth through Runx2 transcriptional inhibition and AKT inactivation, which was regulated by miR-802, suggesting the potential therapeutic value for HCC.

Keywords: Runx2; ZNF521; hepatocellular carcinoma; miR-802; proliferation.

Figure 1

ZNF521 is significantly down-regulated in HCC tissues and cell lines. (A) Relative ZNF521 mRNA expression levels in HCC tissues and matched adjacent nontumor tissues were determined by qRT-PCR. (B) Representative Western blot analysis of ZNF521 expression in the HCC (T) and nontumor tissues (NT) was shown. (C) Representative images of IHC staining of ZNF521 in HCC and adjacent non-tumor tissues. Comparing differences in the expression level of ZNF521 protein (D) between HCC cell lines with the immortalized normal hepatic cell LO2. (E) HCC patients with lower expression of ZNF521 had worse overall survival and disease-free survival. (F) Cases with high/low ZNF521 expression were analyzed. n = three repeats with similar results; *P < 0.05.

Figure 2

ZNF521 inhibited cell proliferation, colony formation and viability in vitro. (A) Hep3B and Huh7 cells that were transfected with different vectors were subjected to western blot for ZNF521 expression. ZNF521 overexpression inhibited cell proliferation (B), colony formation (C) and viability (D) in Hep3B cells, while ZNF521 knockdown promoted proliferation (B), cell colony formation (C) and viability (D) in Huh7 cells. (E) Western blot analysis of protein Cyclin D1 and p21 expression in the presence and absence of ZNF521. *P<0.05. EV, empty vectors. n=three independent experiments; *P<0.05.

Figure 3

ZNF521 inhibited tumor growth in vivo. (A) Tumor growth curve revealed that ZNF521 overexpression significantly inhibited tumor growth in vivo. (B) Tumor nodules were subjected to immunohistochemical staining for Ki-67 assays and (C) TUNEL and quantitative analysis. Scale bar: 70 µM; *P < 0.05.

Figure 4

ZNF521 antagonizes Runx2 transcriptional activity in HCC cells. (A) ZNF521 inhibited Runx2 transcriptional activity. ZNF521 overexpression inhibited Runx2 mRNA (B) and protein (C) expression while ZNF521 knockdown promoted Runx2 mRNA (B) and protein (C) expression. (D) ZNF521-overexpressing Hep3B cells that were transfected with Runx2 overexpression vectors and ZNF521-supressing Huh7 cells that were transfected with Runx2 knockdown vectors were subjected to immunoblotting for Runx2. Runx2 overexpression reversed the inhibitory effects on cell proliferation (E), colony formation (F) and viability (G) of ZNF521-overexpressing Hep3B cells; *P < 0.05.

Figure 5

AKT phosphorylation acts downstream of ZNF521 to mediate the effects on HCC cells. (A) ZNF521 overexpression inhibited AKT phosphorylation while ZNF521 knockdown promoted AKT phosphorylation. AKT phosphorylation inhibitor MK2206 reversed the promotive effects on cell proliferation (B), colony formation (C), viability (D) and related-factors expression (E) of ZNF521-suppressing Huh7 cells; *P < 0.05.

Figure 6

ZNF521 is identified as a direct target of miR-802 in HCC. (A) miR-802 and its putative binding sequence in the 3'-UTR of ZNF521. (B) miR-802 significantly suppresses the luciferase activity that carried wild-type (wt) but not mutant (mt) 3'-UTR of ZNF521. Anti-miR-802 led to a notable increase in the luciferase activity of wt 3'-UTR of ZNF521. (C) qRT-PCR analysis of ZNF521 mRNA expression in Huh7 cells with miR-802 or miR-control vector transfection and Hep3B cells with anti-miR-802 or anti-miR-NC vector transfection. (D) Overexpression of miR-802 reduced the expression of ZNF521 protein in Huh7 cells and knockdown of miR-802 increases the level of ZNF521 protein in Hep3B cells. (E) The expression level of miR-802 in HCC tissues and adjacent non-tumor tissues. (F) A significant inverse correlation between the mRNA levels of ZNF521 and miR-802 was observed in HCC tissues. U6 as the internal control; *P < 0.05.