【作者】 李刚；

【导师】 李湘平；

【作者基本信息】 第一军医大学 ， 耳鼻咽喉科学， 2004， 硕士

【摘要】 目的： 1 探讨能否应用人工合成siRNA诱导RNA干扰，抑制鼻咽癌细胞中EB病毒潜伏膜蛋白1(LMP-1)的表达。 2．观察LMP-1基因沉默对鼻咽癌细胞转移能力的影响。 3．探讨shRNA表达载体诱导RNA干扰技术在LMP-1基因的功能研究及抗转移基因治疗中的应用价值。 方法： 人工合成4条不同序列的双链siRNA，采用脂质体转染法导入鼻咽癌细胞后，根据半定量RT-PCR检测到的LMP-1 mRNA水平变化，筛选出有效的RNA干扰序列。 根据有效干扰序列构建shRNA表达载体，导入EB病毒(+)的鼻咽癌细胞C666-1，筛选出LMP-1基因沉默的亚株。通过观察该业株细胞贴壁生长能力、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4个候选序列中筛选出1有效干扰序列。导入该siRNA后，C666-1细胞中LMP-1 mRNA水平下降90％以上，初次转染后36小时重复转染，能够增强基因抑制效果，延长有效干扰时间。 shRNA表达载体转染后，筛选出LMP一1基因沉默的C666一1亚株，在EBNA一1及GAPDH等无关基因不受影响的情况下，LMP一1的表达受到稳定抑制，达到接近“基因敲除”的效果。 与对照组相比，LMP.1基因沉默的鼻咽癌细胞，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:1.人工合成siRNA诱导RNA干扰，能够有效抑制鼻咽癌细胞C666一1中LMP一1基因的表达。2.shRNA表达载体也能够在鼻咽癌细胞内诱导RNA干扰，并获得对LMP一1基因稳定的抑制。3.LMP一1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不同方面影响鼻咽癌细胞的转移。4.人工诱导RNA干扰抑制LMP一1基因表达，有可能成为鼻姻癌抗转移基因治疗的有效策略。更多还原

【Abstract】 Objective: To evaluate the application of RNA interference for silencing latent membrane protein-l(LMP-l) expression in EBV-positive NPC cell line C666-1. Analysis the inhibiting effects of LMP-1 silencing on NPC cell metastasis. Methods: Four synthesized siRNAs were transfected into C666-1 cell using OligofectamineTMreagent, and LMP-1 inhibition effect was determined by semi-quantitative RT-PCR. DNA vectors were constructed as shRNA vector according to the critical target region of LMP-1. A LMP-1 silenced sub-clone named C611L was isolated by standard calcium phosphate transfection procedure and G418 selection. Then, effect of LMP-1 inhibition on its metastatic properties was evaluated by eelL adhesion, invasion and wound healing assays. Results: The the critical target sequence was identified from the 4 candidates. Single dose of this siRNA caused nearly 90% loss of LMP-1 mRNA in C666-1 cell. The specific inhibition could last for 96 hours following a re-transfectinn. Moreover, shRNA vector also mediated stable RN.Ai against LMP L in C666-1. Compared with control groups, loss of LMP-1 significantly altered cell motility, substratum adhesion and transmembrane invasion ability. Conclusions: These results provided solid testimony thai EBV-encoded latent membrane protein-1 is vulnerable to RNA interference. Stable suppression of LMP-1 by shRNA vector can inhibit metastatic properties of- 4 -NPC cell, which indicated that DNA vector-based RNA interference , as a powerful tool, is benefit to further investigating LMP-1 functions in NPC. It also suggested a novel RNAi-mediated therapeutic strategy for EBV-reLated NPCs.更多还原