干扰ROR1基因表达可抑制骨肉瘤MG-63细胞的上皮-间质转化

Interference of ROR1 gene expression inhibits epithelial-mesenchymal transition of osteosarcoma MG-63 cells

目的:探讨干扰受体酪氨酸激酶样孤核受体1(receptor tyrosine kinase-like orphan receptor 1,ROR 1)基因表达对骨肉瘤MG-63细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响。方法:将特异性靶向ROR 1基因的siRNA(即ROR1-siRNA)转染至ROR1相对高表达的骨肉瘤MG-63细胞中,蛋白质印迹法检测ROR 1基因表达下调情况。然后采用划痕愈合实验及Transwell小室法检测MG-63细胞迁移和侵袭能力的变化,倒置光学显微镜下观察细胞形态的变化。最后,采用蛋白质印迹法和免疫荧光染色法检测MG-63细胞中EMT相关蛋白E-钙黏蛋白(E-cadherin)和波形蛋白(vimentin)以及肿瘤转移相关蛋白锌指E盒结合同源盒蛋白1(zincnger E-box binding homeobox protein 1,ZEB1)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9的表达情况。结果:转染ROR1-siRNA可明显下调骨肉瘤MG-63细胞中ROR1蛋白的表达水平(P<0.05)。下调ROR 1基因表达后,MG-63细胞的迁移和侵袭能力均明显降低(P值均<0.05),细胞形态由间质细胞形向上皮细胞形转变,E-cadherin表达水平明显上调(P<0.05),而vimentin表达水平明显下调(P<0.05),另外ZEB1、MMP-2和MMP-9的表达水平均明显降低(P值均<0.05)。结论:RNA干扰ROR 1基因表达可通过抑制EMT的发生,降低骨肉瘤MG-63细胞的迁移和侵袭能力。

Objective： To investigate the effect of interferencing receptor tyrosine kinase-like orphan receptor 1 （ROR1） gene expression on epithelial- mesenchymal transition （EMT） of osteosarcoma MG-63 cells.Methods： The specific siRNA targeting ROR1 gene （RORI-siRNA） was transfected into osteosarcoma MG-63 cells with the relative high expression of ROR1 protein, then the down-regulation of ROR1 expression was confirmed by Western blotting. The migration and invasion abilities of MG-63 cells were examined by scratch wound healing assay and Transwell chamber assay. The morphological changes of MG-63 cells were observed under an inverted optical microscope. Finally, the expressions of EMT-associated E-cadherin and vimentin, and tumor metastasis-related zinc finger E-box binding homeobox protein 1 （ZEB1）, matrix metalloproteinase 2 （MMP-2） and MMP-9 proteins in MG-63 cells were detected by Western blotting and immunofluoresence staining, respectively. Results： RORI-siRNA transfection significantly inhibited the expression of ROR1 in MG-63 cells （P 〈 0.05）. After down-regulating ROR1 gene expression by RORI-siRNA transfection, the migration and invasion abilities of MG-63 cells were significantly reduced （both P 〈 0.05）, the morphology of MG-63 cells conversed from mesenchymal phenotype to epithelial phenotype, and the expression level of E-cadherin was significantly up-regulated （P 〈 0.05）, while the expression level of vimentin was significantly down-regulated （P 〈 0.05）. Furthermore, the expressions of ZEB1, MMP-2 and MMP-9 in MG-63 cells transfected with ROR1 -siRNA were decreased as compared with those in the untransfected group （all P 〈 0.05）. Conclusion： RNA interference of ROR1 gene expression can reduce the migration and invasion abilities of osteosarcoma MC,-6~ r^ll~ thrnHnh inhihitinn th~ nrrllrr^nr~ nf I：MT