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目的利用RNA干扰技术,以淋巴样增强因子(LEF-1)为靶基因,设计、构建重组体,研究其对人恶性黑素瘤细胞A375中LFF-1表达的抑制作用。方法设计、合成针对LEF-1 mRNA序列的有小发夹结构的正义和反义寡核苷酸,退火后与表达载体Psilencer3．1-H1 neo相连接,经鉴定正确后转染人恶性黑素瘤细胞A375,以G418筛选,获得稳定转染细胞株,分别以逆转录聚合酶链反应(RT-PCR)、蛋白质印迹法(Western blot)和免疫细胞化学方法检测转染细胞中LEF-1 mRNA及蛋白表达水平的改变。结果成功构建了靶向人LEF-1基因的siRNA表达载体,获得稳定转染细胞株,转染细胞LEF-1 mRNA 及蛋白水平明显下调。结论靶向LEF-1的RNA干扰重组体可抑制人恶性黑素瘤细胞A375中LEF-1 表达。
Objective To design and construct recombinants by using RNA interference technology and using lymphoid enhancement factor (LEF-1) as target genes to study their inhibitory effect on LFF-1 expression in human malignant melanoma cells A375. METHODS: The sense and antisense oligonucleotides with small hairpin structure targeting LEF-1 mRNA sequence were designed and synthesized. After annealing, they were ligated to the expression vector Psilencer3.1-H1 neo. After correct identification, they were transfected into human melanin. The tumor cells A375 were screened with G418 to obtain a stable transfected cell line. The LEF-1 mRNA in the transfected cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry. And changes in protein expression levels. Results The siRNA expression vector targeting human LEF-1 gene was successfully constructed, and a stable transfected cell line was obtained. The mRNA and protein levels of LEF-1 were significantly down-regulated in transfected cells. Conclusion LEF-1 targeting RNA interference recombinants can inhibit the expression of LEF-1 in human malignant melanoma cells A375.