siRNA下调AEG-1表达对神经母细胞瘤细胞MMP-9的表达及侵袭能力的影响

Knockdown of AEG-1 mediates matrix metalloproteinase-9 expression and cell invasion in human neuro- blastoma cell lines

目的观察下调Astrocyte elevatedgene-1（AEG-1）基因对神经母细胞瘤细胞中基质金属蛋白酶-9（MMP-9）表达以及细胞侵袭能力的影响。方法根据RNA干扰原理，设计合成AEG-1siRNA。通过荧光定量RT-PCR和Westernblotting观察神经母细胞瘤细胞中AE（2-1基因mRNA和蛋白下调情况；采用Western blotting观察下调AEGl表达对神经母细胞瘤细胞中MMP-9蛋白表达的影响；通过侵袭实验（Transwell法）观察下调AEG-I表达对神经母细胞瘤细胞侵袭能力的影响。结果采用AEG-1siRNA转染神经母细胞瘤细胞，下调细胞内AEG-1的mRNA和蛋白表达（P〈0．05）后MMP-9蛋白表达抑制率分别为63．1％和58．9％。此外下调AEG-1表达使细胞侵袭能力明显减弱（P〈0．05）。结论AEG-1基因下调使神经母细胞瘤细胞中MMP-9的蛋白表达明显减少、活性明显减弱，同时使细胞的侵袭能力降低。由此可见AEG-1在肿瘤细胞的侵袭转移中可能扮演重要角色，将会成为神经母细胞瘤基因治疗的新靶点。

Objective To investigate the effect of astrocyte elevated gene-1 （AEG-1）siRNA induced inhibition of invasion in neuroblastoma cells. Methods A small interference RNA （siRNA） targeting to AEG-1 mRNA （AEG-1 siRNA） was constructed and transfected into neuroblastoma cells with Lipofectamine 2000. A non-specific siRNA （control siRNA） and non-treatment were used as negative control group and blank group. The expression of AEC-1 mRNA was detected by RT-PCR. The proteins of AEG-1 and MMP-9 were detected by Western blotting. Cell invasion after AE@I knockdown was observed by Transwell assay. Results Compared with the control group, the expression of AEG-1 mRNA and protein were significantly decreased in the cells transfeeted with AEG-1 siRNAs （P〈0. 05）. AEG-1 knockdown by siRNA markedly decreased the expression of MMP-9 pro tein by 63. 1 % in neuroblastoma cells and the ability of cell invasion （P〈0. 05）. Conclusions The expression of AEG-1 mRNA is down-regulated by AEG-1 siRNA in neuroblastoma cells. Moreover, knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits the expression of MMP9 protein and cell invasion. Therefore, AEG-1 may play a key role for MMP-9 activity and cell invasion, making it a potential target gene for gene-therapy in neuroblastoma.