| Objective:To explore the expression of Ezrin protein in osteosarcoma tissues and its relationship with clinical pathological characteristics of osteosarcoma.Methods:1. Paraffin specimens including126osteosarcoma and27osteochondroma tissues were collected. The expression of Ezrin protein was detected by immunohistochemistry staining.2. The relationship was analyzed between Ezrin protein expression in osteosarcoma tissues and clinical pathological characteristics such as sex, age, tumor diameter, pathological types, Ennecking stages, location, pulmonary metastasis, and differentiation.Results:1. With the increase of positive rate of Ezrin protein in osteosarcoma tissues (81.74%)was higher than that in osteochondroma tissues (29.63%), and the difference had significance (Χ2=35.627, P<0.01).2. The positive rate of Ezrin protein in osteosarcoma tissues with pulmonary metastasis (97.78%) was significantly increased as compared to that with no pulmonary metastasis (72.84%), and there was significant difference between them (Χ2=15.684, P<0.05). The above indicator in osteosarcoma tissues with low differentiation (94.87%) was elevated when compared to that with middle and high differentiation (75.86%)(Χ2=12.315, P<0.05). However, there was no relationship with sex, age, tumor diameter, pathological types, Ennecking stages and location (P>0.05).Conclusion:The expression of Ezrin protein was obviously inereased in osteosarcoma tissues, and closely correlates with pulmonary metastasis, and differentiation degree. Objective:To explore the role of Ezrin protein in the invasion and metastsis of osteosarcoma by down-regulating the expression of Ezrin protein in osteosarcoma MG-63cells via small interfering RNA (siRNA).Methods:1. MG-63cells were transfected by3Ezrin siRNAs (Ezrin siRNA1, Ezrin siRNA2and Ezrin siRNA3). The expression levels of Ezrin mRNA and protein were measured by fluorescence real time PCR and Western blotting to screen1siRNA with the most inhibitory effect.2. Then, this Ezrin siRNA and Scramble siRNA transfected MG-63cells, repectively,.and we also set up blank control group untransfected by siRNA. Cell proliferation was detected using MTT method.3. The abilities of migration and invasion were analyzed through Transwell assay. Cell adhesive capacity was measured by adhesive experiment.4. The expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9), E-cadherin, N-cadherin, vimentin, Src, phosphorylated-Src (p-Src), Akt and phosphorylated-Akt (p-Akt) were detected through Western blotting.5. Thirty null mice were randomly divided into blank control group (n=10), Scramble siRNA (n=10) and Ezrin siRNA group (n=10). Subsequently, mice in blank control group, Scramble siRNA and Ezrin siRNA group were injected with untransfected MG-63cells, and transfected Scramble siRNA and Ezrin siRNA MG-63cells, respectively, by caudal vein. On day42, all mice were sacrificed, and body weight before and after experiments as well as nodus number of pulmonary surface were compared.Results:1. The inhibitory effects of Ezrin siRNA3on Ezrin mRNA and protein expression were better than Ezrin siRNA1and Ezrin siRNA1(P<0.01). Thus, we selected Ezrin siRNA3to perform the subsequent experiments.2. At24h,48h and72h, the optical density (OD) values were lower in Ezrin siRNA group than blank control and Scramble siRNA groups (P<0.05).3.Transwell migration and invasion experiments demonstrated that the cell numbers of permeated membrane were lower in Ezrin siRNA group than blank control and Scramble siRNA groups (P<0.05). At the same time, Cell adhesive rates were were significantly reduced in Ezrin siRNA group compared with blank control and Scramble siRNA groups (P<0.05).4. In comparison with control group, the expression levels of MMP-2, MMP-9, N-cadherin, vimentin, p-Src and p-Akt were reduce but E-cadherin expression level were increased in Ezrin siRNA group (P<0.01). However, the levels of Src and Akt had no changes (P>0.05).5. After experiments, the mean body weight was significantly increased (P<0.05) but nodus number of pulmonary surface of null mice was significantly decreased in Ezrin siRNA group as compared to blank control and Scramble siRNA groups.Conclusion:1. Downregulation of Ezrin protein expression by siRNA can inhibit the proliferation, invasion and metastsis of MG-63cells.2. The inhibitory effects of Ezrin siRNA on the invasion and metastsis of MG-63cells are associated with downregulation of MMP-2, MMP-9, N-cadherin and vimentin expression, upregulation of E-cadherin, and suppression of Src and Akt phosphorylation. Objective:To observe the effects of baicalein on the proliferation and invasion of MG-63cells as well as the expression and activity of Ezrin, and explore the mechanisms by which baicalein protects against osteosarcoma.Methods:1. MG-63cells were cultured in vitro. Baicalein was diluted into6concentrations by dehydrated alcohol, namely1,2,4,8,16and32μmol/L. Cells were treated with above baicalein for24h,48h and72h, repectively. Cell proliferation was detected using MTT method. At48h after baicalein intervention,50%inhibitory concentration (IC50) was calculated.2. Then, MG-63cells were incubated with dehydrated alcohol (as control group),10and15μmol/L baicalein, repectively. The abilities of migration and invasion were analyzed through Transwell assay.3. Cell adhesive capacity was measured by adhesive experiment.4. The expression of Ezrin mRNA as well as Ezrin and p-Ezrin protein were detected by fluorescence real time PCR and Western blotting.Results:1. With the increase of baicalein concentrations and prolongment of time, inhibitory rates of cell proliferation were gradually increased (P<0.01). IC50was approximately18.35μmol/L at48h after baicalein intervention.2. Transwell migration and invasion experiments showed that the cell numbers of permeated membrane were lower in10and15μmol/L baicalein groups than control group (P<0.05,0.01). Moreover, those were also lower in15μmol/L baicalein group than10μmol/L baicalein group (P<0.05).3. Cell adhesive rates were lower in10and15μmol/L baicalein groups than control group (P<0.05,0.01). Those in15μmol/L baicalein group were significantly reduced compared with10μmol/L baicalein group (P<0.05).4. In comparison with control group, the expression levels of Ezrin mRNA as well as Ezrin and p-Ezrin protein were significantly decreased (P<0.05,0.01). Futhermore, the above indicators were lower in15μmol/L baicalein group than10μmol/L baicalein group (P<0.05).Conclusion:1. Baicalein can inhibit the proliferation of MG-63cells in time and concentration-dependent manners.2. Baicalein downregulates the expression of Ezrin mRNA as well as Ezrin and p-Ezrin protein. |
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