TrKA -siRNA抑制乳腺癌细胞NF-κB的表达并促细胞凋亡

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

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

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

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/Abstract

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.

章菊陈昌杰刘臣彪杨清玲滕凤猛. TrKA -siRNA抑制乳腺癌细胞NF-κB的表达并促细胞凋亡[J]. 基础医学与临床, 2009, 29(10): 1059-1064.

Ju ZHANG; Chang-jie CHEN; Chen-biao LIU; Qing-ling YANG; Feng-meng TENG. TrKA -siRNA inhibits the expression of NF-κB and promote the apoptosis of breast cancer[J]. Basic & Clinical Medicine, 2009, 29(10): 1059-1064.