RNA干扰技术抑制人恶性黑素瘤细胞A375中淋巴样增强因子mRNA及蛋白表达

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。

Inhibition of lymphoid enhancer factor-1 expression in human malignant melanoma cell line A375 by RNA interference method

Objective To investigate the inhibition of lymphoid enhancer factor-1 (LEF-1) expression in human malignant melanoma cell line A375 by RNA interference method. Methods Sense and antisense oligonucleotides with hairpin structures, targeted specifically at LEF-1 mRNA, were designed, synthesized, then linked to the expression vector psilencer3.1-H1 neo after annealing. After identification, the re-combinant psilencer3.1-H1/LEF-1 siRNA was used to transfect the cultured A375 cells by a liposome-medi-ated method. The cells expressing the recombinant RNA was detected by G418 screening. The mRNA and protein levels were detected by RT-PCR, Western blotting and immunocytochemistry, respectively. Results The expression vector psilencer3.1-H1/LEF-1 siRNA was successfully constructed, and its stable expression in cell clones was achieved. The mRNA and protein levels of LEF-1 were both down-regulated in the trans-fected cells. Conclusion The recombinant of psilencer3.1-HI/LEF-1 siRNA can inhibit the mRNA and protein expression of LEF-1 in A375 cells.