中华皮肤科杂志 ›› 2008, Vol. 41 ›› Issue (9): 594-597.

• 论著 • 上一篇    下一篇

短发卡RNA对黑素瘤B16F10细胞BAG-1基因表达的抑制作用

宋亚丽 陈浩 刘毅 陈佳 薛燕宁 曾学思 孙建方   

  1. 南京医科院皮研所 南京中国医学科学院中国协和医科大学皮肤病研究所 南京市中国医学科学院皮肤病研究所 南京医科院皮研所 南京医科院皮研所 南京医科院皮研所
  • 收稿日期:2007-11-02 修回日期:2007-11-21 发布日期:2008-09-15
  • 通讯作者: 宋亚丽 E-mail:songyali@medmail.com.cn

Silencing BAG-1 gene by eukaryotic expression vector containing short hairpin RNA in mouse melanoma B16F10 cells

  

  • Received:2007-11-02 Revised:2007-11-21 Published:2008-09-15

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摘要： 目的 构建针对小鼠BAG-1（Bcl-2-associated athanogene 1）基因的短发卡RNA（shRNA）真核表达质粒，探讨其对小鼠黑素瘤B16F10细胞BAG-1基因表达的抑制作用。方法 以小鼠BAG-1 mRNA编码区作为RNA干扰靶点，构建shRNA真核表达质粒pRNAT-U6.1/Neo-BAG-1，应用lipofectaminTM 2000转染小鼠黑素瘤B16F10细胞，设阴性对照组（转染阴性对照质粒Neg）和空白对照组（未转染），转染后48 h开始G418筛选。转染1个月后采用RT-PCR、蛋白质印迹检测BAG-1基因的表达。结果 酶切及测序结果证实，重组质粒pRNAT-U6.1/Neo-BAG-1构建成功。质粒在B16F10细胞的转染效率为20% ~ 30%。阴性对照组细胞BAG-1基因表达与空白对照组无差异，shRNA质粒处理组细胞BAG-1基因表达较空白对照组显著下降（P < 0.05），表达抑制率在mRNA和蛋白水平分别为77% ± 4%和62% ± 2%。结论 针对小鼠BAG-1基因的shRNA真核表达质粒pRNAT-U6.1/Neo-BAG-1能高效特异地抑制小鼠黑素瘤B16F10细胞BAG-1基因的表达。

关键词: shRNA, BAG-1, 黑色素瘤

Abstract: Objective To construct the eukaryotic expression plasmids of short hairpin RNA （shRNA） specific for mouse Bcl-2-associated athanogene 1 （BAG-1） and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16F10 cells. Methods Plasmids named pRNAT-U6.1/Neo-BAG-1, were designed and constructed to target the mouse BAG-1 mRNA coding region. LipofectaminTM 2000 was used to transfect plasmids into B16F10 cells. Negative plasmid-transfected and untransfected B16F10 cells served as negative and blank controls respectively. Forty-eight hours following transfection, G418 was used to select the resistant cells. The mRNA and protein expression of BAG-1 gene was measured by reverse transcription-PCR and Western blot respectively about 1 month after the transfection. Results The eukaryotic expression plasmids, pRNAT-U6.1/Neo-BAG-1, were constructed, and verified by restriction enzyme digestion and DNA sequencing. The transfection rate in B16F10 cells was 20%-30%. Compared with the blank control, the mRNA and protein expression of BAG-1 in B16F10 cells was significantly inhibited by BAG-1 shRNA （both P < 0.05）, and the inhibition rates were （77 ± 4）% and （62 ± 2）%, respectively. Conclusions These results indicate that the eukaryotic expression vectors containing shRNA against BAG-1 gene, pRNAT-U6.1/Neo-BAG-1, are successfully constructed, and can significantly inhibit the expression of BAG-1 gene in mouse melanoma B16F10 cells.

Key words: small hairpin RNA, BAG-1, melanoma