基础医学与临床 ›› 2009, Vol. 29 ›› Issue (10): 1059-1064.

• 研究论文 • 上一篇    下一篇

TrKA -siRNA抑制乳腺癌细胞NF-κB的表达并促细胞凋亡

章菊 陈昌杰 刘臣彪 杨清玲 滕凤猛   

  1. 蚌埠医学院临床检验诊断实验中心 蚌埠医学院生化与分子生物学教研室
  • 收稿日期:2008-09-04 修回日期:2008-12-22 出版日期:2009-10-20 发布日期:2009-10-20
  • 通讯作者: 陈昌杰

TrKA -siRNA inhibits the expression of NF-κB and promote the apoptosis of breast cancer

Ju ZHANG, Chang-jie CHEN, Chen-biao LIU, Qing-ling YANG, Feng-meng TENG   

  1. Reseach Center of Clinical Laboratory Science, Beng bu Medical College Department of Biochemistry & Molecular Biology, Beng bu Medical College
  • Received:2008-09-04 Revised:2008-12-22 Online:2009-10-20 Published:2009-10-20
  • Contact: Chang-jie CHEN,

摘要: 目的 探讨TrKA基因表达的变化对NF-κBp65核蛋白的表达和细胞凋亡的影响。方法 构建TrKA特异小干扰RNA(siRNA)表达载体,重组体经测序鉴定正确后转染乳腺癌MCF-7细胞。G418(geneticin)筛选稳定表达TrKA siRNA干扰细胞株,命名为TrKA-siRNA。荧光实时定量RT-PCR,Western blot和免疫组化法检测TrKA基因的表达,Western blot检测NF-κBp65核蛋白的表达,流式细胞术检测细胞凋亡。结果 成功构建 TrKA-siRNA 表达载体,TrKA mRNA和蛋白水平分别下调74.7%和80.5%(P<0.05)。阳性对照 GAPDH 基因表达水平下降85.0%(p<0.05);NGF作用2 h后,MCF-7组细胞NF-κBp65核蛋白表达明显增加,而TrKA-siRNA组细胞NF-κBp65核蛋白改变不显著。与MCF-7组相比,TrKA- siRNA组细胞凋亡明显增加(p<0.05),NGF对其凋亡无促进作用。结论 有效阻断TrKA基因的表达能抑制NF-κB抗凋亡途径的活化,从而增加了乳腺癌MCF-7细胞的凋亡。此作用可能不依赖于外源性的NGF。

Abstract: Objective Investigate the effect of the change of TrKA gene on the expression of neucleoprotein NF-κBp65 and apoptosis. Method To construct the expression vector of TrKA small interfering RNA,The recombinant was transfected into MCF-7 cells. the stable cell line expressing TrKA small interfering RNA were selected by G418. The mRNA and protein of TrKA were tested by real-time PCR,Western-blot and Immunohistochemistry.The change of neucleoprotein NF-κBp65 was detected by WB, Flow cytometry was used to observe the cell apoptosis. Result The expression vector of TrKA-siRNA was successfully constructed.The mRNA and protein of TrKA was decreased by 74.7% and 80. 5% respectively ( P < 0. 01).The positive control of GAPDH was decreased by 85.0% (p<0.05). The change of neucleoprotein NF-κBp65 was dramatical after treat the MCF-7 cells with NGF for 2 hours.but not to the siRNA cells. Compared with MCF-7 group,the apoptosis of the group of TrKA-siRNA increase obviously(p<0.05),but NGF can not enchance the effect. Conclusion Interfering the expression of TrKA gene effectively,could inhibit the activity of neucleoprotein NF-κBp65,increase the apoptosis of breast cancer cell MCF-7 independent of NGF.