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目的 :探讨沉默受体相互作用蛋白激酶4(receptor interacting protein kinase4,RIPK4)基因表达对骨肉瘤U-2OS细胞上皮-间质转化(epithelialmesenchymal transition,EMT)的影响。方法 :采用脂质体法将特异性针对RIPK4基因的si RNA转入骨肉瘤U-2OS细胞,构建RIPK4基因沉默表达的U-2OS细胞系,随后采用蛋白质印迹法检测RIPK4表达的变化。采用Transwell小室法检测沉默RIPK4基因表达后对U-2OS细胞迁移和侵袭能力的影响;倒置光学显微镜下观察细胞形态的变化。最后,釆用蛋白质印迹法检测U-2OS细胞中EMT标志物E-钙黏蛋白(E-cadherin)和波形蛋白(vimentin)的表达水平。结果:蛋白质印迹法检测结果显示,RIPK4-si RNA转入U-2OS细胞后,RIPK4蛋白的表达水平明显下调(P<0.05);RIPK4-si RNA转染组U-2OS细胞的迁移和侵袭能力明显下降(P值均<0.05);U-2OS细胞的形态由间质形向上皮细胞形转变。RIPK4-si RNA转染组细胞中E-cadherin的表达水平明显上调(P<0.05),而vimentin的表达水平明显下调(P<0.05)。结论 :沉默RIPK4基因表达可抑制骨肉瘤U-2OS细胞EMT的发生,并降低细胞的迁移和侵袭能力。
OBJECTIVE: To investigate the effect of receptor interacting protein kinase 4 (RIPK4) gene expression on the epithelialmesenchymal transition (EMT) in human osteosarcoma U-2OS cells. Methods: The si RNA specific to RIPK4 gene was transfected into U-2OS cells by lipofectamine to construct U-2OS cell line with RIPK4 silencing. The expression of RIPK4 protein was detected by Western blotting. The effect of silencing RIPK4 gene expression on the migration and invasion ability of U-2OS cells was detected by Transwell chamber method. The changes of cell morphology were observed under inverted light microscope. Finally, Western blotting was used to detect the expression levels of EMT markers E-cadherin and vimentin in U-2OS cells. Results: The results of Western blotting showed that the expression of RIPK4 protein was significantly down-regulated after RIPK4-si RNA transfection into U-2OS cells (P <0.05). The migration and invasion of U-2OS cells in RIPK4-si RNA transfected group (P <0.05). The morphology of U-2OS cells changed from the interstitial to epithelial cells. The expression of E-cadherin in RIPK4-si RNA transfected cells was significantly up-regulated (P <0.05), while the expression of vimentin was significantly down-regulated (P <0.05). Conclusion: Silencing the expression of RIPK4 can inhibit EMT in U-2OS cells and reduce the migration and invasion of osteosarcoma cells.