沙门菌介导骨桥蛋白siRNA对Tca 8113细胞体外生长的影响

Effect of attenuated Salmonella carrying osteopontin siRNA on viability and invasiveness of Tca 8113 cells

目的:研究应用减毒沙门菌感染性转基因骨桥蛋白siRNA(OPNsiRNA)抑制Tca 8113细胞生长和转移的可行性和作用。方法:人工合成OPNsiRNA,同时,合成无义ControlsiRNA作为对照,进行以下平行试验;合成siRNA,分别构建表达载体pIRES2-EGFP,转染减毒沙门菌SL7207,获得的重组菌SL-OPNsiRNA和SL-ControlsiRNA分别体外感染Tca 8113细胞,观察细胞感染率及对细胞增殖、凋亡、迁移和侵袭力的影响,同时以Western印迹检测转基因OPNsiRNA对细胞内源性OPN、基质金属蛋白酶-2(MMP-2)和尿激酶型纤溶酶原激活因子(uPA)蛋白表达的影响。采用SPSS15.0软件包进行组间t检验。结果:重组减毒沙门菌感染Tca 8113的效率可达80.0%以上,两者之间无显著差异(P>0.05);OPNsiRNA可显著抑制Tca 8113细胞的生长和诱导凋亡;感染后24h,细胞OPNsiRNA和Control-siRNA的迁移指数分别为0.56±0.10和0.82±0.05,侵袭力指数分别为0.53±0.13和0.83±0.06,2组之间均存在显著差异(n=12,P<0.01);同时,OPNsiRNA感染可显著降低细胞内OPN、MMP-2和uPA蛋白的表达量。结论:携带OPN-siRNA的减毒沙门菌可有效通过细菌感染方式,转入舌癌细胞,发挥抗肿瘤生长和转移的作用。

PURPOSE：To explore the effectiveness of Osteopontin siRNA（OPNsiRNA） on viability and invasiveness of Tca 8113 by attenuated Salmonella typhimurium.METHODS： Synthesised OPNsiRNA and nonsense ControlsiRNA were separately constructed into expression vector pIRES2-EGFP,and were transferred into attenuated Salmonella SL7207.The tongue cancer cell line Tca 8113 was infected by the recombinant Salmonella SL7207,named SL-OPNsiRNA and SL-ControlsiRNA respectively.The infection rate,growth,apoptosis,migration and invasion were observed while the protein expressions of OPN,MMP-2 and uPA were detected by Western blot.SPSS15.0 software package was used for statistical analysis.RESULTS： The infection rates of the recombinant Salmonellas both were more than 80.0% without significant difference（P0.05）.The growth was significantly inhibited and the apoptosis was significantly induced after infection of SL-OPNsiRNA compared with SL-ControlsiRNA.After 24h-infection of SL-OPNsiRNA and SL-ControlsiRNA,the migration index was 0.56±0.10 and 0.82±0.05（n=12,P0.01）,the invasion index were 0.53±0.13and 0.83±0.06（n=12,P0.01）,respectively,and the intra-cell proteins of OPN,MMP-2 and uPA were also significantly down-regulated by OPNsiRNA compared with ControlsiRNA.CONCLUSIONS：OPNsiRNA could be effectively transferred into tongue cancer cells through attenuated Salmonella,and inhibited caner cell growth and metastasis.