DNA载体介导RNA干扰抑制LMP-1基因对鼻咽癌细胞转移能力的影响

目的探讨应用DNA载体介导RNA干扰技术,抑制鼻咽癌细胞中EB病毒潜伏膜蛋白1（LMP-1）表达的可能性,观察LMP-1基因沉默对鼻咽癌细胞转移能力的影响。方法将能转录出干扰RNA的DNA模板插入pAVU6＋27质粒,构建能够介导RNA干扰的发夹状干扰RNA（shRNA）表达载体。采用磷酸钙共沉淀法,将质粒导入EB病毒（＋）的鼻咽癌细胞C611,筛选LMP-1基因沉默的克隆,通过观察其贴壁生长能力、基底膜穿透能力和移动能力的变化,分析LMP-1基因沉默对鼻咽癌细胞转移能力的影响。结果在shRNA表达载体有效转染的C611细胞中,LMP-1基因的表达受到稳定抑制。肿瘤细胞转移实验表明,LMP-1基因沉默的C611细胞,贴壁生长能力提高,基底膜穿透能力和细胞移动能力均明显下降。结论shRNA表达载体能够在鼻咽癌细胞内介导RNA干扰,并获得对LMP-1基因稳定的抑制。LMP-1基因可能通过贴壁生长能力、基底膜穿透能力和细胞移动能力等,影响鼻咽癌细胞的转移。

Inhibition of EBV-encoded LMP-1 by DNA-based RNA interference affects metastatic ability of nasopharyngeal carcinoma cells

Objective To evaluate the application of DNA vector-based RNA interference for silencing LMP-1 expression in EBV-positive nasopharyngeal cancer (NPC) C611 cells and analyze the inhibiting effects of LMP-1 silencing on metastatic property of NPC cells. Methods DNA vectors with LMP-1 shRNA template were transfected into C611 cells. After G418 selection, a LMP-1 silenced sub-clone named C611L was isolated. Effect of LMP-1 inhibition on its metastatic properties was evaluated by cell adhesion, invasion and wound healing assays. Results shRNA vector mediated stable RNAi against LMP-1 in C611 cells. Loss of LMP-1 significantly altered cell motility, substratum adhesion and transmembrane invasion ability. Conclusion Stable suppression of LMP-1 induced by shRNA vector can inhibit metastatic properties of NPC cells.