TrKA-siRNA抑制乳腺癌细胞系MCF-7 NF-κB的表达并促细胞凋亡

TrKA-siRNA inhibits the expression of NF-κB and promote apoptosis of breast cancer cell line MCF-7

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TrKAsiRNA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基因和蛋白的表达,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)。神经生长因子(NGF)作用2h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。

Objective Investigate the effect of TrKA variation on the expression of neucleoprotein NF-κB P65 and apoptosis. Methods To construct the expression vector of TrKA small interfering RNA, the recombinant was trans-fected 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-κB P65 was detected by WB, Flow cytometry was used to observe the cell apoptosis. Results The expression vector of TrKA-siRNA was successfully constructed. The mRNA and protein of TrKA were decreased by 74.7% and 80.5% respectively （P〈0.01）. The change of neucleoprotein NF-κB P65 was dramatical after treating MCF-7 cells with NGF for 2 hours. Compared with MCF-7 group, the apoptosis of TrKA-siRNA group increased significantly（P〈0.05）, but NGF failed to enchance the effect. Conclusion Interfering the expression of TrKA gene effectively, may inhibit the activity of neucleoprotein NF-κB P65 and increase the apoptosis of breast cancer cell MCF-7 independent of NGF.