中华皮肤科杂志 ›› 2006, Vol. 39 ›› Issue (11): 625-628.

• 论著 • 上一篇    下一篇

短发卡RNA对人黑素瘤细胞BRAF基因的沉默作用

韩永智, 孙建方, 周武庆, 曾学思, 姜祎群   

  1. 中国医学科学院、中国协和医科大学皮肤病研究所
  • 收稿日期:2006-01-16 出版日期:2006-11-15 发布日期:2006-11-15
  • 通讯作者: 孙建方, email:sunjanf@jlonline.com E-mail:email;sunjanf@jlonline.com

Silence of BRAF gene in human melanoma cells by plasmid mediated shRNA

HAN Yong-zhi, SUN Jian-fang, ZHOU Wu-qing, ZENG Xue-si, JIANG Yi-qun   

  1. Department of Dermato-histopathology, Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, China
  • Received:2006-01-16 Online:2006-11-15 Published:2006-11-15

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摘要： 目的构建BRAF基因序列特异性的短发卡RNA(shRNA)质粒载体,检测其对人黑素瘤细胞BRAF基因的干扰作用。方法设计2条BRAF基因序列特异性的shRNA编码序列和1条非特异性阴性对照序列,插入质粒pGenesil-1,双酶切和测序鉴定。用构建成功的3条质粒转染人黑素瘤细胞株A375和M14,分别采用RT-PCR和蛋白印迹检测其BRAF基因的mRNA和蛋白表达。结果所设计的shRNA序列被正确插入质粒pGenesil-1。非特异性对照质粒neg对黑素瘤细胞BRAF基因表达不产生干扰,2条序列特异性质粒braf1和braf2抑制了BRAF基因mRNA和蛋白的表达,其中braf1干扰效果最为明显,最高抑制率可达90%。结论质粒介导的shRNA可成功干扰人黑素瘤细胞BRAF基因的表达,其抑制时间可持续至少1个月。

关键词: 黑色素瘤, RNA干扰, BRAF基因, shRNA

Abstract: Objective To construct the short hairpin RNA (shRNA)-expressing plasmid vectors specific for BRAF gene,and to test their effects in BRAF knockdown in human melanoma cell lines.Methods Two pairs of specific BRAF shRNA oligoes and a pair of randomly synthesized non-specific shRNA oligo were synthesized and inserted into plasmid pGenesil-1.Their fidelity was confirmed by double endonuclease digestion and sequencing.The constructed plasmids were transfected into human melanoma cell lines A375 and M14.The expression of BRAF mRNA and BRAF protein were detected by RT-PCR and Western blotting,respectively.Results The designed shRNA oligoes were precisely cloned into the plasmid pGenesil-1.The expression of BRAF mRNA and protein were down-regulated by specific plasmid braf 1 and braf 2,except to non-specific plasmid neg.The plasmid braf 1 was more effective,reducing BRAF gene expression by 90 per cent.Conclusions Plasmid mediated shRNA could successfully knockdown BRAF expression in human melanoma cells,and the suppression of the gene expression could maintain for 1 month at least.

Key words: Melanoma, RNA interference, BRAF, shRNA