| Aims:To study the effects of Ezrin gene on biological behaviors in cervical cancer and explore the molecular mechanism of Ezrin in cervical cancer progression.Methods:1. Experiment in vitro:Western blotting was used to detect the protein expression of Ezrin in HeLa, SiHa, C33 A, CaSki cervical cancer cell lines, cervical cancer and normal cervical tissue. Then, the protein expression of Ezrin was detected in Ezrin-siRNA-transfected cells by Western blotting and immunofluorescence staining. Meanwhile, the cell proliferation was detected by colony formation assay. Series of molecular techniques, such as wound-healing and transwells assay were used to determin the migration and invasion in HeLa and SiHa cells after transfect with si-Ezrin and si-control. Furthermore, Western blot and immunofluorescence staining were used to detect the expression of epithelial marker E-cadherin, CK-18 and the mesenchymal markers Vimentin, MMP-2 and β-catenin in Ezrin-siRNA-transfected and control cells. At last, Western blotting was used to determine the effects of Ezrin depletion on the activities of Akt, S6,4EBP-1.2. Experiments in vivo:"Artificial air sac" methods and silicone ring for the inoculating carder in CAM was used to monitor invasion of cervical cancer after knockdown of Ezrin gene.3. Additionally, immunohistochemical staining of Ezrin protein was performed on uterine cervical cancer specimens from 235 patients. For comparison, 239 cases of cervical intraepithelial neoplasia (CIN),17 cases of cervical glandular intraepithelial neoplasia (CGIN) and 52 normal cervix samples were also included. qRT-PCR was performed on fresh tissues to detect Ezrin mRNA expression levels. HPV infection statuses were genotyped by oligonucleotide microarray, and 10-year survival rates were calculated using the Kaplan-Meier method for 109 cervical cancer patients.Results:1. Experiments in vitro:Ezrin protein was highly expressed in four cervical cancer cells and three cervical cancer tissues compared to normal tissue by Western blotting. Ezrin protein expression was decreased by transfect siRN A in HeLa and SiHa cells. The cultured cells showed tight cell-cell adhesion and significant cell polarity by phase-contrast microscopy observation in Ezrin-siRNA-transfected compare to si-control cells. Cells transfected with si-Ezrin formed fewer colonies than cells transfected with si-control (P<0.01). Moreover, knocking down Ezrin expression significantly decreased the migration and invasiveness of cervical cancer cells compared with the control group, as measured by the wound healing and transwell assays. Furthermore, the expression of epithelial marker E-cadherin, CK-18 were increased, but the mesenchymal markers Vimentin, MMP-2 and β-catenin were decreased by Western blotting and immunofluorescence staining in Ezrin-siRNA-transfected cells. At last, p-Akt was decreased in si-Ezrin-transfect HeLa cells.2. Experiments in vivo:The CAM assay shown that cells transfected with si-Ezrin showed less invasion into the CAM than si-control cells.3. Furthermore, the strongly positive rate of Ezrin was significantly higher in cervical cancers than in CIN, CGIN, and normal cervical epithelia. Ezrin overexpression was closely related with poor differentiation, late stage, and lymph node metastasis. Additionally, Ezrin overexpression was associated with lower 10-year survival rate for patients with early stage cervical cancer, but not for patients with advanced stage.Conclusions:Down-regulation of Ezrin significantly inhibited proliferation, migration, and invasion of uterine cervical cancer cells through inhibition of EMT. This action may be related to the PI3K/Akt pathway. Also, the overexpression of Ezrin protein might predict the poor prognosis of patients with cervical cancer, and Ezrin might be an independent effective biomarker for prognostic evaluation of cervical cancers. |
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