中华皮肤科杂志

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短发卡RNA抑制黑素瘤细胞BRAFV600E突变基因的研究

陈浩1, 韩永智2, 周武庆1, 曾学思1, 孙建方1   

  1. 1. 中国医学科学院、中国协和医科大学皮肤病研究所, 南京, 210042;
    2. 广州省人民医院
  • 收稿日期:2007-01-11 出版日期:2007-06-15 发布日期:2007-06-15
  • 通讯作者: 孙建方,email:sunjanf@jlonline.com E-mail:sunjanf@jlonline.com
  • 基金资助:
    江苏省六大高峰人才基金资助

In vivo and in vitro suppression of BRAFV600E in human melanoma cells by short hairpin RNA

CHEN Hao1, HAN Yong-zhi2, ZHOU Wu-qing1, ZENG Xue-si1, SUN Jian-fang1   

  1. Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences&Peking Union Medical College, Nanjing 210042, China
  • Received:2007-01-11 Online:2007-06-15 Published:2007-06-15

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摘要： 目的 探讨BRAFV600E突变基因在黑素瘤发展过程中的作用。方法 用构建成功的质粒载体转染人黑素瘤细胞株A375,并建立动物模型来检测其在体内的干扰作用。分别采用RT-PCR和蛋白质印迹法检测体内外BRAF基因的mRNA和蛋白表达。结果 特异性短发卡RNA在体内外均可抑制人黑素瘤细胞BRAF基因mRNA和蛋白的表达,使其蛋白表达量分别减少62%和90%。在动物模型中使黑素瘤细胞的成瘤性下降,可以抑制肿瘤增长缓慢,抑瘤率达到62%。结论 BRAFV600E突变基因在黑素瘤的发展中有一定的作用。

关键词: shRNA, 黑色素瘤, 实验性, 基因, BRAF, RNA干扰

Abstract: Objective To investigate the role of BRAFV600E in the development of melanoma,as well as in vivo and in vitro suppression of BRAFV600E by short hairpin RNA(shRNA).Methods Previously constructed shRNAs targeted against the BRAF gene were used.Three plasmids,including two BRAF-specific shRNA plasmids,braf 1 and braf 2,and a negative control plasmid,were constructed and transfected into human melanoma cell line A375.A xenograft nude mouse model was used to examine the inhibitory effect ofshRNA in vivo.The expressions ofBRAF mRNA and protein were detected by RT-PCR and Western blotting,respectively.Results The specific shRNA inhibited the in vivo and in vitro expression of BRAF mRNA and protein;protein level was decreased by 62% in vivo and 90%/n vitro.In the mouse model,the tumorigenicity was decreased by the specific shRNA and the growth of tumor was inhibited by 62%.Conclusion The specific shRNAs suppress the mRNA and protein expression of BRAFV600E,and this gene may play an important role in the development of melanoma.

Key words: shRNA, Melanoma, experimental, Genes, BRAF, RNA interference