The Novel Zinc Finger Protein 587B Gene, ZNF587B, Regulates Cell Proliferation and Metastasis in Ovarian Cancer Cells in vivo and in vitro

Abstract

Background: The zinc finger protein 587B (ZNF587B) is a novel cisplatin-sensitive gene that was identified in our previous research by using a genome-scale CRISPR-Cas9 knockout library in ovarian cancer (OC) cell lines. ZNF587B belongs to the C2H2-type zinc finger protein (ZFP) family. Many ZFP protein could inhibit tumor development and malignancy. However, the function of ZNF587B remains unknown.

Methods: Quantitative PCR (qPCR) was utilized to compare ZNF587B mRNA expression levels in OC and normal ovarian cell lines. The small interfering RNA (siRNA) and full-length ZNF587B eukaryotic expression plasmid were constructed and transfected into OC cells later. Colony formation, 5-ethynyl-2'-deoxyuridine (EdU) assay, transwell assay, and xenograft experiment were conducted to evaluate the effect of ZNF587B on OC cells.

Results: ZNF587B was downregulated by approximately 43% and 17% in the OC cell lines SKOV3 and A2780, respectively, compared with that in the normal ovarian cell line IOSE80. Overexpression of ZNF587B reduced cell proliferation, colony formation, migration, and invasion, which could be reversed by knockdown of ZNF587B via siRNA. Xenograft experiments also confirmed that ZNF587B could suppress tumor growth. Survival data of OC patients in the SurvExpress database showed that with respect to overall survival, low-risk patients grouped by the prognostic index had a higher expression of ZNF587B and a better prognosis than high-risk group (HR = 1.77, 95% CI: 0.55-0.70, p = 0.023). Moreover, overexpression of ZNF587B promoted OC cells apoptosis when pretreated with cisplatin.

Conclusion: ZNF587B is a novel potential tumor suppressor of OC and may be a therapeutic target for OC.

Keywords: C2H2-type zinc finger protein; ZNF587B; metastasis; ovarian cancer; proliferation.

Figure 1

ZNF587B was downregulation in OC cell lines and modulation of ZNF587B regulated OC cell clonality. The expression level of ZNF587B was examined by qPCR in OC cells A2780 and SKOV3 compared with normal ovarian cells IOSE80 (A). The knockdown efficiency was detected by qPCR (B). ZNF587B overexpression efficiency was detected by qPCR (C). Consumption of ZNF587B contributed to cell clonality and overexpression of ZNF587B impaired colony forming ability in A2780 (E) and SKOV3 (F) cells. Cloning efficiency was measured by statistics counting clone number (D). Results were representative of at least three independent experiments showing similar results. All the data above were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.0.001.

Figure 2

Modulation of ZNF587B affected OC cell proliferation. ZNF587B knockdown promoted cell proliferation, while overexpression of ZNF587B significantly inhibited cell proliferation measured by EDU assay (A) A2780 and (B) SKOV3. Cell numbers counting was subjected on ImageJ analysis. Results were representative of at least three independent experiments showing similar results. All the data above were presented as mean ± SD. **P < 0.01; ***P < 0.0.001; ****P < 0.0001.

Figure 3

Modulation of ZNF587B regulated migration and invasion of OC cells. ZNF587B knockdown significantly increased cancer cellular migration and invasion, while ZNF587B overexpression significantly decreased cellular migration and invasion in A2780 (A) and SKOV3 (B) cells. Quantitative data of migration cell number was shown in (C) after 48 h in OC cells. Quantitative data of invasive cell number was shown in (D) after 48 h in OC cells. Results were representative of at least three independent experiments showing similar results. All the data above were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.0.001; ****P < 0.0001.

Figure 4

Overexpression of ZNF587B inhibited tumor growth in vivo. Schematic diagram of tumorigenesis in nude mice and collected tumors were showed in (A). The body weight of mice was shown before inoculated and removed tumors (B). Tumor growth curves of control A2780 cells and ZNF587B stable-overexpression A2780 cells (C). Tumor were collected and weighed in pc3.1 group and pc3.1-ZNF587B group (D). Tumor volume of xenografts was evaluated as 0.5 LW.² *P < 0.05.

Figure 5

Correlation between ZNF587B expression and cisplatin response in ovarian cell line. Groups transfected with either siRNA-ZNF587B or pc3.1-ZNF587B were subjected to treatment with cisplatin in concentrations at 3 μM, 6 μM, 9 μM, 12 μM, 15 μM, 18 μM (A). Representative plots of Annexin V/7-AAD apoptosis assay in A2780 siRNA-ZNF587B, vector, pc3.1-ZNF587B, and pc3.1 groups with or without cisplatin treatment by flow cytometry analysis (B). Percentage of apoptotic cells was shown in histogram (C). Each assay was performed at least three times on biological replicates. *P < 0.05; **P < 0.01.

Figure 6

OC patients with upregulation ZNF587B were associated with better prognosis and survival. OC patients were divided into low-risk and high-risk groups by prognostic index (A). Box plot showing the ZNF587B gene expression level by risk groups in OV-AU-ICGC OC-Serous cystadenocarcinoma-June 2016 dataset generated by ICGC (from the SurvExpress database) (B). The Kaplan–Meier survival curve comparing the ZNF587B high-expression population (green) and ZNF587B low-expression population (red) of OC patients was created from the SurvExpress database (C).