Table 1.
Primer Sequences used for qRT-PCR.
Figure 1.
Immunohistochemical staining of ezrin in human tongue SCCs and noncancerous tissues.
(A) Ezrin expression was low in noncancerous tissues (magnification 40× and 200×). (B) Ezrin was highly expressed in tongue squamous cell carcinomas (magnification 40× and 200×). Ezrin immunoreactivity was apparent in the membrane of normal tongue epithelium, whereas positive staining for ezrin was primarily in the cytoplasm or in the membrane and cytoplasm of tongue squamous cell carcinoma cells.
Table 2.
Association between ezrin expression and clinico-pathologic variables in 79 tongue tissue.
Figure 2.
Correlations between ezrin expression and the indices of Ki-67 and apoptosis in human tongue SCC tissues.
(A) The Ki-67 index was 33.0±19.3% in tissues with low ezrin expression and 48.1±15.0% in tissues with high ezrin expression. Significant differences were observed in correlation between ezrin expression and the Ki-67 index (P = 0.0003). (B) There was no significant correlation between ezrin expression and apoptotic indices (P = 0.5776).
Figure 3.
The expression of ezrin in HSC-3 tongue SCC cells after siRNA treatment.
(A) Ezrin mRNA expression was analyzed using real-time PCR after RNAi. The inhibition of ezrin mRNA expression was clearly observed in ezrin siRNA-transfected HSC-3 cells compared with that in HSC-3 control cells (0.10±0.03 vs 1.06±0.21; P<0.05). (B) Ezrin protein expression was detected by western blotting after RNAi. The expression of ezrin decreased dramatically in ezrin siRNA-transfected HSC-3 cells.
Figure 4.
Cell cycle changes after RNAi treatment.
The proportion of HSC-3 cells in the S phase decreased from 50.8±1.6% to 36.6±0.4% after RNAi (P = 0.0018). The proportion of cells in the G0/G1 phase also increased from 37.7±4.9% to 54.1±0.5% after RNAi (P = 0.0018). Apoptosis was not detected.
Figure 5.
Effects of ezrin downregulation on HSC-3 cell growth.
The growth of ezrin siRNA-transfected HSC-3 cells decreased in a time-dependent manner. (24 h: P = 0.6727; 48 h: P = 0.5314; 72 h: P = 0.0122; 96 h: P = 0.0065).
Figure 6.
Cells were wounded by scratching with a pipette tip, and mitomycin (3 mg/mL) was added to the medium for 1 h to inhibit the proliferation of cancer cells. Cells were subsequently incubated with DMEM for 48 h. Cells were photographed using phase-contrast microscopy. HSC-3 cell migration was reduced by ezrin siRNA treatment.
Figure 7.
(A) Matrigel-coated Transwell chambers were used to analyze cell invasion. Cells that infiltrated through the filter were fixed and stained, and representative fields were photographed. (B) The cells were quantified by counting under a light microscope. Compared with the HSC-3 control cells, the ezrin siRNA-transfected HSC-3 cells exhibited significantly decreased invasiveness (1969.8±126.6 vs 564.7±111.8; P<0.05).
Figure 8.
NSC- and ezrin siRNA-transfected HSC-3 cells were labeled with an antibody against actin. Ezrin depletion of HSC-3 cells led to reduced ezrin protein levels. This downregulation was associated with the loss of protrusions (arrows).
Figure 9.
Western blot analyses of E-cadherin, N-cadherin, β-catenin, and Rho family proteins in HSC-3 cells.
The expression of E-cadherin and β-catenin was increased and the expression of N-cadherin was decreased in ezrin siRNA-transfected HSC-3 cells compared with that in the NSC-transfected cells. There was no significant difference in the total protein levels of RhoA, Rac1 and Cdc42.