超声及超声微泡联合shRNA质粒对survivin基因的沉默效应

Ultrasound and microbubbles mediated shRNA transfection silences survivin gene expression

目的:研究超声及超声微泡联合短发夹状RNA(shRNA)干扰技术对宫颈癌细胞(Hela)survivin基因的沉默效应。方法:构建3个靶向survivin基因的shRNA真核表达质粒(pSIREN/S1/S2/S3)和无关序列质粒(pSIREN/con),对培养的Hela细胞行超声及超声微泡联合处理(US+SO)或脂质体转染,以空白细胞组、单纯超声辐照组、pSIREN/S1组、超声辐照+pSIREN/S1组为对照,应用蛋白质印迹和RT-PCR检测survivin蛋白表达及mRNA转录水平的变化。结果:酶切、测序分析证实重组质粒构建成功。RT-PCR和蛋白质印迹法表明,3种pSIREN/S均可抑制Hela细胞内survivin mRNA和蛋白质的表达(P<0.05),而pSIREN/con无此作用,以pSIREN/S3的抑制效果最显著(P<0.05);US+SO组的mRNA和蛋白表达水平的抑制率分别为(87.04±1.80)%和(85.94±1.02)%,均显著高于其他各组(P<0.05)。结论:survivin可将作为宫颈癌基因治疗的理想靶标,超声及超声微泡联合shRNA干扰技术可显著沉默靶基因survivin的表达。

Objective：To study the effect of the association of ultrasound and micmbubble with short hairpin RNA （shRNA） on the expression of survivin mRNA and protein in eervieal cancer （HeLa） eell lines. Methods： Three survivin-shRNA expression vectors （pSIREN/S1/ S2/S3） were constructed and introduced into HeLa eells. A corresponding negative control vector was constructed （pSIREN/eon）. The shRNA vectors were added to Hela cells transfected by Lipofeetamine^TM（P ＋ L） or followed by ultrasound exposure with SonoVue microbubble （US ＋ SO）. Blank control group, pSIREN/S1 plasmid only, ultrasound exposure only, pSIREN/S1 and ultrasound exposure were used as eontrols, respectively. The expressions of survivin mRNA and protein were detected by RT-PCR and western blot analysis, respectively. Results： Three recombinant plasmids were suceessfully constructed. The plasmids were showed to inhibit the expression of survivin gene mRNA and protein efficiently and speeifieally exeept for the pSIREN/eon, while pSIREN/S3 was significantly lower than other groups （ P 〈 0.05）. After transfected with pSIREN/S3 for 48 h, the inhibition ratios of survivin mRNA and protein in US ＋ SO group [ （87.04 ± 1.80）%, （85.94±1.02）% ] were markedly higher than in any other groups （ P 〈 0.05）. Conclusion：It is suggested that survivin could be regarded as an ideal target for new anticancer intervention of cervical cancer. The association of ultrasound and microbubbles with shRNA could effectively silence the expression of target survivin gene.