Zinc Finger Protein 521, Negatively Regulated by MicroRNA-204-5p, Promotes Proliferation, Motility and Invasion of Gastric Cancer Cells

Abstract

Objective: This study aims to investigate the expression, role, and detailed mechanism of microRNA-204-5p and zinc finger protein 521 in gastric cancer.

Methods: Immunohistochemistry was adopted to detect the expressions of zinc finger protein 521 in 82 cases of gastric cancer tissues. Western blot was used to detect the expressions of zinc finger protein 521 in gastric cancer cells and adjacent cells. Moreover, the correlation between zinc finger protein 521 and the prognosis of patients were also evaluated. Cell Counting Kit 8 assay and colony formation assay were performed to figure out the impact of zinc finger protein 521 on the proliferation of gastric cancer cells. By conducting flow cytometry, the effect of zinc finger protein 521 on the apoptosis of gastric cancer cells was determined. The scratch wound healing assay and transwell invasion assay were carried out to determine the effect of zinc finger protein 521 on regulating the motility and invasion of gastric cancer cells. Ultimately, the targeting relationship and interaction between microRNA-204-5p and zinc finger protein 521 were verified by real-time polymerase chain reaction, Western blot, and dual luciferase reporter gene assay.

Results: Compared with adjacent cells, zinc finger protein 521 was highly expressed in gastric cancer cells, which was related to TNM stage (P = .0388), tumor size (P = .0168), and local lymph node metastasis (P = .0024). Overexpressed zinc finger protein 521 can promote the proliferation, migration, and invasion of gastric cancer cells and inhibit the apoptosis. Zinc finger protein 521 is a target gene of microRNA-106-5p, and there was a negative correlation between the expression of zinc finger protein 521 and microRNA-204-5p.

Conclusion: Zinc finger protein 521 can arrest the apoptosis and enhance the proliferation, migration, and invasion of gastric cancer cells via regulating microRNA-204-5p. Our study may provide novel clues for the treatment of patients with gastric cancer.

Keywords: ZNF521; gastric cancer; miR-204-5p.

Figure 1.

The expression of ZNF521 was increased in GC cells and tissues. A, Immunohistochemical images of GC tissues (left) and adjacent tissues (right). B, Immunohistochemistry was used to detect the difference of ZNF521 expressions between GC tissues and adjacent tissues (P < .001). C, Western blot was carried out to detect the expressions of ZNF521 in GC cell lines (MKN-28, SGC-7901, BGC-823, MKN-45, NCI-N87, and AGS) and normal gastric mucosal cells (GES-1). *, ** and *** refer to P < .05, P < .01, and P < .001, respectively. GC indicates gastric cancer; ZNF521, zinc finger protein 521.

Figure 2.

Comparison of prognostic indications between patients with overexpression of zinc finger protein 521 (ZNF521) and those with low expression of ZNF521. A, Comparison of the overall survival (OS) between patients with overexpression of ZNF521 and those with low expression of ZNF521. B, Comparison of the first progression (FP) between patients with overexpression of ZNF521 and those with low expression of ZNF521. C, Comparison of the postprogression survival (PPS) between patients with overexpression of ZNF521 and those with low expression of ZNF521.

Figure 3.

Zinc finger protein 521 potentiates the proliferation but impedes the apoptosis of GC cells. A, The transfection efficiency was detected by Western blot. B, Colony formation assay was conducted to monitor the proliferation of AGS (left) with overexpressed ZNF521 and MKN-28 (right) with knockdown ZNF521. C, Flow cytometry was carried out to detect the apoptosis of AGS (left) with overexpressed ZNF521 and MKN-28 (right) with knockdown ZNF521. *** refers to P < .001. GC indicates gastric cancer; ZNF521, zinc finger protein 521.

Figure 4.

Zinc finger protein 521 promotes the motility, migration, and invasion of GC cells. A, The motility of AGS (left) with overexpressed ZNF521 and MKN-28 (right) with knockdown ZNF521 detected by scratch wound healing assay. B, The migration of AGS (left) with overexpressed ZNF521 and MKN-28 (right) with knockdown ZNF521 determined by transwell assay. C, The invasion of AGS (left) with overexpressed ZNF521 and MKN-28 (right) with knockdown ZNF521 monitored by transwell assay. D, The expressions of Bcl-2, BAX, E-cadherin, and N-cadherin in GC cells were detected by Western blot. GC indicates gastric cancer; ZNF521, zinc finger protein 521.

Figure 5.

Target relations between miR-204-5p and ZNF521 were verified by dual luciferase reporter gene assay. A, There was a binding site between miR-204-5p and ZNF521. B, The luciferase activity of wild type and mutant type ZNF521 was compared. C, MicroRNA-204-5p was negatively correlated with ZNF521 (Spearman correlation, P <.0001, R = −0.6584). D, The expressions of ZNF521 in AGS and MKN-28 transfected with miR-204-5p were detected by Western blot. E, The expressions of ZNF521 mRNA in AGS transfected with miR-204-5p mimics and MKN-28 transfected with miR-204-5p inhibitors were detected by PCR. *** indicates P < .001. miR-204-5p indicates microRNA-204-5p; mRNA, messenger RNA; PCR, polymerase chain reaction; ZNF521, zinc finger protein 521.