反义Snail转录因子对骨肉瘤细胞增殖和转移能力的影响

Inhibitory effect of antisense snail expression plasmid on proliferation,invasion and migration of osteosarcoma MG-63

目的:探讨反义Snail转录因子对人骨肉瘤细胞株MG-63增殖和转移的影响。方法:用脂质体转染法将高转移性骨肉瘤细胞株MG-63分别转染反义Snail质粒(pGenesil-snailAS)和空载体质粒(pGenesil)。G418筛选阳性细胞,获得稳定转染的阳性克隆。采用免疫组化、Western blot及半定量逆转录-聚合酶链反应(RT-PCR)检测不同组MG-63骨肉瘤细胞中Snail表达;体外细胞运动实验及重组细胞基底膜(Matrigel)侵袭实验了解细胞转移潜能的改变;以噻唑蓝(MTT)比色法和克隆形成实验观察细胞增殖。结果:MG-63细胞内Snail表达明显,转染反义Snail质粒后,Snail表达明显被抑制。Snail表达下降的同时伴随着体外细胞增殖、黏附和侵袭力的抑制。结论:骨肉瘤细胞中存在Snail蛋白及mRNA的高表达,抑制Snail mRNA的表达对骨肉瘤细胞的侵袭、运动和增殖有抑制作用,可能为骨肉瘤的基因治疗提供新的靶点。

Objective To investigate the inhibitory effect of antisense snail expression plasmid on invasion and proliferation of osteosarcoma MG-63 cell line in vitro. Methods pGenesil-snail-AS and pGenesil were transfected into M G-63 cells using LipofectamineTM 2000 reagent,the stably transfected clones were screened using G418 sulfate. MG-63 ceils were divided into three groups ： cells transfected with pGenesil-snail-AS, cells transfected with pGenesil and normal control group. Snail mRNA and protein were detected using Western blot, immunohistochemical assay and RT-PCRanalysis. The inhibitory effects of antisense-snail on MG-63 cells were determined by MTT, transwellmotility assay, matrigel invasion assay, and colony formation assay. Results Cell growth inhibition was found in MG-63 ceils transfected with pGenesil-snail-AS compared with normal control and cells transfected with pGenesil. The motility activity and the invasion activity of antisense-snail-transfectant cells were significantly decreased compared to those of normal cells and pGenesil-transfected cells. There were no differences between normal control cells and pGenesil- transfected cells. Conclusions The present data indicate that snail is a potential target for cancer gene therapy, snail inhibition can block invasion, metastasis, and proliferation of human osteosarcoma.