Ezrin Promotes the Proliferation, Migration, and Invasion of Ovarian Cancer Cells
Objective The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms. Methods We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells. Results The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial–mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation. Conclusion Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.
- Epithelial ovarian cancer /
- Ezrin /
- Proliferation /
- Invasiveness /
- RhoA /
- Stress fiber
Figure 1. Effects of ezrin on cell proliferation in ovarian carcinoma cells
(A) The protein levels of ezrin were analyzed via Western blot analysis in IOSE80, CaOV3, and SKOV3 cells. (B) SKOV3 and CaOV3 cells were transfected with siEzrin or FLAG-ezrin and their respective negative controls for 24 h. The efficiency of the knockdown or upregulation of ezrin was verified via Western blot analysis. (C) Cell growth was determined via MTT assay after ezrin overexpression or knockdown in SKOV3 and CaOV3 cells. Data are presented as mean ± standard deviation, n ≥ 3, **P < 0.01. (D) Colony formation assays were performed on the upregulation and downregulation of ezrin in SKOV3 and CaOV3 cells.
Figure 2. Effects of ezrin on cell migration in ovarian carcinoma cells
(A) Cell migration assays were performed on the upregulation and downregulation of ezrin in SKOV3 and CaOV3 cells. (B) Statistical results of cell migration efficiency. Data are presented as mean ± standard deviation, n ≥ 3, *P < 0.05, **P < 0.01. (C) Representative images of wound healing assay conducted in OVCA cells (SKOV3 and CaOV3) transfected with pCMV-FLAG-ezrin (FLAG-ezrin) or pCMV-FLAG empty vector (FLAG-vector).
Figure 3. Effects of ezrin on cell invasion in ovarian carcinoma cells
(A) Cell invasion assay and (B) quantification showed invasiveness of OVCA cells (SKOV3 and CaOV3) transfected with pCMV-FLAG-ezrin (FLAG-ezrin), pCMV-FLAG empty vector (FLAG-vector), siNC, siEzrin. Data are presented as mean ± standard deviation, n ≥ 3, *P < 0.05, **P < 0.01.
Figure 4. Effects of ezrin on EMT in ovarian carcinoma cells
SKOV3 and CaOV3 cells were transfected with pCMV-FLAG-ezrin (FLAG-ezrin), pCMV-FLAG empty vector (FLAG-vector), siNC, anti-ezrin siRNA for 24 h and then harvested for Western blot analysis and RT-qPCR. (A) Western blot analysis showed the protein level of ezrin and EMT-associated markers (E-cadherin and vimentin). RT-qPCR showed the mRNA level of vimentin (B) and E-cadherin (C) in SKOV3 and CaOV3 cells. *P < 0.05, **P < 0.01.
Figure 5. Ezrin regulates RhoA GTPase activity
Analysis of RhoA activation levels after the overexpression or knockdown of ezrin in SKOV3 (A) and CaOV3 cells (B) through the immunoprecipitation of their GTP active forms. The ratio of RhoA-GTP and Rho A is shown below. Analysis of Rac1 activation levels after the overexpression or knockdown of ezrin in SKOV3 (C) and CaOV3 (D) through the immunoprecipitation of their GTP active forms.
Figure 6. Ezrin stimulates the formation of stress fibers
(A) SKOV3 cells and (B) CaOV3 cells were transfected with either pCMV-FLAG-ezrin (FLAG-ezrin) or pCMV-FLAG empty vector (FLAG-vector) prior to being treated with or without C3 transferase (2 ng/mL). FLAG was detected as described in the Materials and Methods Section (green). F-actin was visualized using Alexa Fluor 568-conjugated phalloidin. Bar: 10 μm.
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