siRNA干扰骨形态发生蛋白2对肝癌SMMC7721细胞迁移和侵袭能力的抑制作用

siRNA-BMP-2 inhibits migration and invasion of human hepatoma cell line SMMC7721

目的设计并化学合成针对骨形态发生蛋白2(bone morphogenetic proteins,BMP-2)的siRNA分子片段,转染人肝癌SMMC7721细胞,观察其对SMMC7721细胞迁移和侵袭能力的影响。方法设计并化学合成针对BMP-2的3个siRNA分子序列,阳离子脂质体法瞬间转染SMMC7721细胞,运用半定量逆转录聚合酶链反应(RT-PCR)和Western印迹法检测细胞中BMP-2在mRNA水平和蛋白水平的变化,体外侵袭实验检测转染后细胞侵袭能力的变化。实验共分6组,①正常对照组;②空白对照组;③阴性对照组;④～⑥分别为BMP-2-siRNA-A、BMP-2-siRNA-B、BMP-2-siRNA-C转染组。结果RT-PCR和Western blot显示3对特异性BMP-2-siRNA转染肝癌细胞SMMC7721之后,其BMP-2的基因和蛋白表达均受到抑制,以siRNA-B抑制效果最明显[(0.32±0.07)vs(0.92±0.14),(0.37±0.10)vs(0.89±0.17),P<0.01]。体外侵袭实验显示转染后肝癌细胞数明显低于未转染组和阴性对照组[分别为(6.48±3.62)、(23.72±3.24)、(22.38±3.84),P<0.05]。结论BMP-2与肝癌的侵袭转移潜能相关,siRNA-BMP-2能明显抑制肝癌细胞SMMC7721的BMP-2表达,从而抑制肝癌细胞的迁移和侵袭能力。

Objective To design and prepare siRNAs targeting bone morphogenetic protein 2 （BMP-2） gene and to observe its effect on the migration and invasion of SMMC7721 cells. Methods Three specific siRNAs targeting BMP-2 were designed and synthesized, and then transiently transfected into SMMC7721 cells via cathodolyte fiposome transfeetion method. Reverse transcriptase PCR and Western blotting were used to detect BMP-2 expression at mRNA and protein levels. The migration and invasion ability of cancer cell were assessed by cell scratch assay and in vitro invasion assay respectively. There are 6 groups, Group Ⅰ ： Normal control group ; Group Ⅱ ： Control group ; Group Ⅲ ： Negative control group ; Group Ⅳ, Ⅴ, Ⅵ ： transfected with BMP-2-siRNA-A,BMP-2-siRNA-B, BMP-2-siRNA-C, respectively. Results After all the 3 specific BMP-2- siRNA （A, B, C） were transfected into SMMC7721 cells, the expression of BMP-2 mRNA and protein were significantly inhibited, with BMP-2-siRNA-B having the highest inhibitory rate [ （0.32 ± 0.07 ） vs （0.92 ±0. 14）, （0.37 ±0.10） vs （0.89 ±0.17）, P 〈0.01 ]. The invasion assay demonstrated that the number of hepatoma cancer cells in transfection group was significantly lower than that in untransfected group, blank con- trol group and negative control group [ （ 6.48 ± 3.62） vs （23.72 ± 3.24）, （22.38 ± 3.84）, P 〈 0. 05 ], which suggested that silencing BMP-2 gene obviously decreased the migration and invasion ability of hepatoma cancer ceils. Conclusion BMP-2 is correlated with the migration and invasion potential of hepatoma cells. The siRNA of BMP-2 gene effectively inhibits the mRNA and protein expression of BMP-2, and then restrain the migration and invasion ability of the cells.