靶向IGF-1R的siRNA增强肺癌细胞对Trail诱导凋亡敏感性的研究  

The study of IGF-1R siRNA enhancing sensibility of lung cancer cell to Trail induced apoptosis

作　　者：刘黎明[1] 胡俊锋[1] 张永[1] 夏雪梅[1] 宫蓓蕾[1] 曹大龙 

机构地区：[1]蚌埠医学院第一附属医院呼吸内科,安徽蚌埠233004 [2]蚌埠市第一人民医院呼吸内科,安徽蚌埠233000

出　　处：《中华全科医学》2016年第12期1987-1989,1993,共4页Chinese Journal of General Practice

基　　金：安徽省教育厅科研基金(KJ2009B222Z)

摘　　要：目的研究IGF-1R特异RNA干扰后肺腺癌细胞A549增殖和凋亡的改变以及其对Trail的敏感性。方法化学合成IGF-1R特异siRNA转录模板,与表达载体连接,转染大肠杆菌扩增后提取质粒,酶切鉴定和DNA测序分析。转染A549细胞后筛选稳定表达IGF-1R-siRNA细胞株,FQ RT-PCR、Western blot和免疫组织化学检测IGF-1R基因表达。MTT法和流式细胞仪测定Trail作用前后转染和非转染A549细胞增殖活性和凋亡率。结果成功构建IGF-1R-siRNA表达载体并筛选出稳定表达IGF-1R-siRNA单克隆细胞。和空白对照相比IGF-1R蛋白和IGF-1RmRNA表达分别下降79.01%(0.17±0.06 vs.0.81±0.15)(P<0.01)和76.05%(1.36±0.26vs.5.68±0.45)(P<0.01)。免疫组织化学显示IGF-1R表达降低。Trail作用于IGF-1R-siRNA的A549,细胞增殖速度降低(P<0.05);凋亡率明显增加(P<0.01)。结论 IGF-1R-siRNA表达载体具有RNA干扰作用。IGF-1R表达下降后A549细胞增殖速度减慢,对Trail诱导的细胞凋亡敏感性增加。Objective To investigate the changes of the proliferation and apoptosis of lung adenocarcinoma cell A549 to IGF-1R specific RNA interference and its sensitivity to Trail. Methods The IGF-1R specific siRNA transcription template was chemically synthesized and connected to the siRNA expression vector. The restructured expression vector was transfected into E. coli. Amplified vector was extracted. Enzyme digestion and DNA sequencing were used to prove that the expression vector was successfully constructed. Stable expression of IGF-1R-siRNA 3.549 cell lines were screened by G418. FQ RT-PCR, Western blot and immunohistochemistry were used to detect inhibitory effect. Before and after the Trail treatment, MTT and flow eytometry were used to test proliferation and apoptosis of A549 cells. Results DNA sequencing results proved the successful construction of IGF-1R-siRNA expression vector. The selected monoclonal ceils with stable expression of IGF-1R-siRNA decreased IGF-1R mRNA and protein expression by 79.01% （0. 17 ± 0.06 vs. 0.81 ± 0.15 ,P 〈 0.01 ） and 76.05% （ 1.36 ± 0.26 vs. 5.68 ± 0.45, P 〈 0.01 ） than that of control A549 cells. Immunohistochemistry also showed a lower expression of IGF-1R. After treatment of Trail on A549 with IGF-1R-siRNA, the cell proliferation rate decreased （ P 〈 0.05 ） ; the apoptosis rate was obviously increased （ P 〈 0.01 ）. Conclusion The constructed IGF-1R-siRNA expression vector can decrease the expression of IGF-1R gene in A549 cells. IGF-1R-siRNA can effectively inhibit the proliferation and enhance the Trail induced apoptosis in lung cancer cell A549.

关 键 词：人胰岛素样生长因子受体1 肿瘤坏死因子相关凋亡诱导配体 RNA干扰 A549细胞 肺肿瘤 

分 类 号：R734.2[医药卫生—肿瘤] R730.54[医药卫生—临床医学]