Forkhead box protein C1 promotes cell proliferation and invasion in human cervical cancer

Abstract

Increasing evidence has demonstrated that aberrant forkhead box protein C1 (FOXC1) expression contributes to tumorigenesis in multiple types of malignant tumor. However, the clinical significance and biological roles of FOXC1 in cervical cancer remain unknown. The expression levels of FOXC1 were examined in human cervical cancer tissues and cells using reverse transcription‑quantitative polymerase chain reaction, immunohistochemistry and western blotting. Furthermore, high FOXC1 expression was significantly associated with advanced clinical stages, a high degree of malignancy and a poor outcome. FOXC1 silencing inhibited cell growth and enhanced cell apoptosis. Knockdown of FOXC1 markedly suppressed cell migration and invasion in vitro, and resulted in downregulation of phosphorylated‑RAC‑α serine/threonine‑protein kinase, proto‑oncogene c‑Myc and B‑cell lymphoma 2. In conclusion, these data indicated that upregulation of FOXC1 contributed to the development of cervical cancer by increasing the growth and motility of the cervical cancer cells, thereby worsening the disease progression in these patients.

Keywords: forkhead box protein C1; cervical cancer; proliferation; migration.

Figure 1.

Upregulated FOXC1 expression in cervical cancer tissues and cell lines. (A) The relative mRNA expression levels of FOXC1 in cervical cancer tissues and control samples, detected by reverse transcription-quantitative polymerase chain reaction. (B) Representative images of immunohistochemistry analyses of FOXC1 protein expression in cervical cancer tissues and adjacent noncancerous tissues (magnification, ×200). (C) The relative expression of FOXC1 in cervical cancer cell lines CaSki, HeLa, ME-180, and SiHa and the NC104 cell line. (D) Western blotting assays of the endogenous FOXC1 protein in cervical cancer cell lines and the immortalized cervical epithelial cell line. The expression levels of FOXC1 were normalized against the expression level of GAPDH. **P<0.01 vs. NC104. FOXC1, forkhead box protein C1.

Figure 2.

Kaplan-Meier survival curves. Kaplan-Meier curves for survival time in patients with cervical cancer, divided according to FOXC1 expression, with significantly shorter survival times for patients with high FOXC1 expression compared with those with low FOXC1 expression. FOXC1, forkhead box protein C1.

Figure 3.

FOXC1 silencing inhibits cervical cancer cell growth in vitro. (A) Western blotting was performed to confirm the effective knockdown of FOXC1. (B) Effect of FOXC1 on Hela and SiHa cell proliferation was measured by Cell Counting Kit-8 assay. (C) Apoptosis was detected by Annexin V-FITC and PI double staining and analyzed by flow cytometry when FOXC1 was silenced. *P<0.05, **P<0.01 vs. shRNA control. FOXC1, forkhead box protein C1; FITC, fluorescein isothiocyanate; PI, propidium iodide; shRNA, short hairpin RNA.

Figure 4.

Knockdown of FOXC1 decreases migratory and invasive abilities of cervical cancer cells. (A) Wound-healing capacity of Hela, SiHa and CaSki cells transduced with control shRNA or FOXC1 shRNA. (B) Matrigel-coated Transwell cell invasion assays of control cells and FOXC1 shRNA stably transduced cervical cancer cells. (C) p-AKT, c-Myc, and Bcl-2 protein levels were downregulated by FOXC1 shRNA in Hela, SiHa and CaSki cells. **P<0.01 vs. shRNA control. shNRA, short hairpin RNA; FOXC1, forkhead box protein C1; p, phosphorylated; AKT, RAC-α serine/threonine-protein kinase (AKT); c-Myc, proto-oncogene c-Myc; Bcl-2, apoptosis regulator B cell lymphoma-2.