Silencing of hypoxia-inducible factor-1α promotes thyroid cancer cell apoptosis and inhibits invasion by downregulating WWP2, WWP9, VEGF and VEGFR2

Abstract

Adaptation to hypoxia is an important process physiologically and pathologically. Hypoxia-inducible factor-1α (HIF-1α) participates in the cancer biology of numerous endocrine tumors, including their proliferation and differentiation. In the present study, the hypothesis that HIF-1α promotes tumorigenesis in thyroid cancer via upregulating angiogenesis-associated markers is investigated. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to examine the expression of HIF-1α in thyroid cancer cell lines, and to detect the expression of WW domain containing E3 ubiquitin protein ligase (WWP)2, WWP9, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in MZ-CRC-1 and TT thyroid cancer cells. Cell proliferation was measured using a Cell Count Kit-8. Cell apoptosis and cell cycle was assessed by flow cytometry. Cell invasive ability was examined by Matrigel transwell analysis. RT-qPCR and western blot analyses demonstrated that the mRNA and protein expression levels of HIF-1α were significant higher in MZ-CRC-1 and TT thyroid cancer cells than in another three thyroid cancer cells (P<0.01). HIF-1α knockdown cells demonstrated inhibition of cell proliferation and invasion, arrested cell cycle at the G1 phase, and induction of cell apoptosis. The protein expression levels of WWP2, WWP9, VEGF and VEGFR2 were decreased in HIF-1α knockdown MZ-CRC-1 and TT cells. In conclusion, HIF-1α may be important in cell apoptosis and invasion of thyroid cancer cells, likely through regulating WWP2, WWP9, VEGF and VEGFR2 expression.

Keywords: angiogenesis; hypoxia; hypoxia-inducible factor-1α; invasion; thyroid cancer.

Figure 1.

Expression of HIF-1α in thyroid cancer cell lines and knockdown by shRNA. (A) mRNA and (B) protein expression of HIF-1α in thyroid cancer cell lines TT, TPC-1, FTC-133, SW579 and MZ-CRC-1. Reverse transcription-quantitative polymerase chain reaction and western blot analysis identified a significant decrease in HIF-1α expression in MZ-CRC-1 cell (C) mRNA and (D) protein levels and TT cell (E) mRNA and (F) protein levels with stable knockdown of HIF-1α. **P<0.01 vs (A and B) FTC-133 cells or (C-F) control groups. HIF-1α, hypoxia-inducible factor-1α; shRNA, small hairpin RNA.

Figure 2.

Effects of HIF-1α on cell proliferation and cell cycle in vitro. (A) Cell Count Kit-8 analysis identified an inhibition of cell proliferation in MZ-CRC-1 and TT cells. (B) Flow cytometry analysis identified an arrest of cell cycle in MZ-CRC-1 and TT cells. **P<0.01 vs control groups. HIF-1α, hypoxia-inducible factor-1α; shRNA, small hairpin RNA; M, mitotic phase; S, synthesis phase.

Figure 3.

Effects of hypoxia-inducible factor-1α on cell apoptosis in vitro. Flow cytometry analysis was performed to determine the apoptotic rate of MZ-CRC-1 and TT cells. **P<0.01 vs. the vector group. shRNA, small hairpin RNA.

Figure 4.

Effects of hypoxia-inducible factor-1α on cell invasion in vitro. Martrigel transwell analysis was performed to determine the invasion of MZ-CRC-1 and TT cell (magnification, ×200). **P<0.01 vs. the vector group. shRNA, small hairpin RNA.

Figure 5.

Effects of hypoxia-inducible factor-1α (HIF-1α) on WWP2, WWP9, VEGF and VEGFR2 expression in MZ-CRC-1 and TT cells. MZ-CRC-1 cell (A) mRNA and (B) protein expression markers with stable knockdown of HIF-1α. TT cell (C) mRNA and (D) protein expression markers with stable knockdown of HIF-1α. **P<0.01 vs. the vector group. WWP, WW domain containing E3 ubiquitin protein ligase; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2.