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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 synthesize a siRNA molecule fragment directed against bone morphogenetic proteins (BMP-2), and to transfect human hepatocellular carcinoma SMMC7721 cells to observe its effect on migration and invasion of SMMC7721 cells. Methods Three siRNA sequences targeting BMP-2 were designed and synthesized. SMMC7721 cells were transiently transfected by cationic liposomes. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of BMP- 2 mRNA and protein levels in vitro, in vitro invasion assay to detect changes in cell invasive ability. The experiment was divided into 6 groups, ① normal control group; ② blank control group; ③ negative control group; ④ ~ ⑥ BMP-2-siRNA-B, BMP-2-siRNA- Dyeing group. Results RT-PCR and Western blot showed that BMP-2 gene and protein expression were inhibited by 3 pairs of specific BMP-2-siRNA siRNA transfected SMMC7721 cells after transfection with siRNA-B [(0.32 ± 0.07 ) vs (0.92 ± 0.14), (0.37 ± 0.10) vs (0.89 ± 0.17), P <0.01]. In vitro invasion assay showed that the number of hepatocellular carcinoma cells after transfection was significantly lower than that of untransfected cells and negative control cells [(6.48 ± 3.62), (23.72 ± 3.24) and (22.38 ± 3.84, P <0.05] respectively. Conclusion BMP-2 is associated with the invasion and metastasis potential of hepatocellular carcinoma. SiRNA-BMP-2 can significantly inhibit the expression of BMP-2 in hepatocellular carcinoma SMMC7721 and thus inhibit the migration and invasion ability of hepatoma cells.