WTAP is a prognostic marker of high-grade serous ovarian cancer and regulates the progression of ovarian cancer cells

Abstract

Background: The Wilms' tumor suppressor WT1 is reported to work in a range of physiological processes at both transcriptional and posttranscriptional level. WT1-associating protein (WTAP), a nuclear protein co-localized with splicing factors, also plays a vital role in cellular function and cancer progression. However, little is known about the role of WTAP in ovarian cancer and the underlying mechanism.

Materials and methods: To evaluate the expression of WTAP, multiple means were applied in clinical tissues, including immunohistochemistry, quantitative reverse transcriptase PCR (qRT-PCR), and Western blot. Two representative ovarian cancer cell lines (3AO and SKOV3) were used to assess the malignant influence of WTAP on proliferation, apoptosis, and migration. To explore its function, WTAP was additionally down-regulated by lentivirus.

Results: High expression of WTAP in high-grade serous ovarian carcinoma (HGSOC) predicted a shorter overall survival (P<0.01). Furthermore, WTAP expression was higher in HGSOC, compared with that in normal ovary group (P<0.01), benign ovarian tumor group (P<0.01), and non-HGSOC group (P<0.05). In HGSOC, high expression of WTAP was significantly related with the lymph node metastasis (P<0.05). In ovarian cancer cell lines, cell proliferation and migration were considerably reduced after WTAP was down-regulated, while apoptotic rate was increased. Moreover, the effect of WTAP in 3AO and SKOV3 might be relevant with MAPK and AKT signaling pathways.

Conclusion: WTAP is highly expressed in HGSOC, and indicates a worse survival outcome. Therefore, it is highly possible that WTAP has a prognostic implication in the patients of HGSOC. In addition, WTAP down-regulation also plays a tumor suppressor role in 3AO and SKOV3 cell lines.

Keywords: WTAP; diagnosis; ovarian cancer; tumor suppression.

Figure 1

Overall survival and disease-free survival curves of HGSOC patients based on WTAP expression levels. (A) The overall survival curve of patients with high WTAP expression (red line) and with low WTAP expression (blue line). (B) The disease-free survival curve of patients with high WTAP expression (red line) and with low WTAP expression (blue line). Abbreviations: HGSOC, high-grade serous ovarian carcinoma; HR, hazards ratio; TPM, transcripts per million.

Figure 2

Immunohistochemistry, Western blot and qRT-PCR results of WTAP expression in different ovarian tissues. (A) Representative WTAP immunohistochemistry staining in different types of ovarian tissues (×400): (a) normal ovary; (b) serous cystadenoma; (c) borderline serous cystadenoma; (d) mucinous cystadenocarcinoma; (e) endometrioid carcinoma; (f) clear-cell carcinoma; (g) low-grade serous ovarian cancer; (h) high-grade serous ovarian cancer. (B, C) Protein expression of WTAP in ovarian tissues. (D) Relative mRNA expression of WTAP in ovarian tissues. *P<0.05, **P<0.01. Abbreviation: ns, non significant.

Figure 3

WTAP regulates cellular proliferation and apoptosis. (A) Protein level of WTAP knockdown efficiency in 3AO and SKOV3 cell lines was detected by Western blot. (B) mRNA level of WTAP knockdown efficiency was detected by qRT-PCR. (C) The influences of WTAP knockdown on cell proliferation were confirmed using the CCK-8 assay. (D) The representative picture of colony formation assay, and the quantification of colonies per well. (E) The representative results of the impact of WTAP knockdown on cell apoptosis, and the quantification of apoptotic rates. (F) The changes of apoptosis-related proteins after down-regulation of WTAP. *P<0.05, **P<0.01. Abbreviation: OD, optical density.

Figure 4

WTAP regulates cellular migration in SKOV3 cell line. (A, C) The effect of WTAP knockdown on SKOV3 migration was detected by transwell assay and scratch wound-healing assay. (B) The results of the transwell assay were quantified. (D) The expression changes of EMT related biomarkers after down-regulation of WTAP. **P<0.01. Abbreviation: EMT, epithelial–mesenchymal transition.

Figure 5

Signaling pathways related to the change of WTAP expression. The effect of WTAP knockdown on the activation of AKT, ERK, JNK, and p38 in 3AO and SKOV3 cell lines.