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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 proliferation and apoptosis of lung adenocarcinoma A549 cells after IGF-1R specific RNA interference and its sensitivity to Trail. Methods IGF-1R specific siRNA transcriptional template was chemically synthesized and ligated with the expression vector. The plasmid was amplified after amplification by E. coli and identified by restriction enzyme digestion and DNA sequencing. The A549 cells were transfected with IGF-1R-siRNA stably expressing cell lines, and the expression of IGF-1R gene was detected by FQ RT-PCR, Western blot and immunohistochemistry. MTT assay and flow cytometry were used to determine the proliferation and apoptosis of transfected and non-transfected A549 cells. Results IGF-1R-siRNA expression vector was successfully constructed and monoclonal cells stably expressing IGF-1R-siRNA were screened out. The expression of IGF-1R protein and IGF-1R mRNA decreased by 79.01% (P <0.01) and 76.05% (P <0.01) compared with the blank control group (P <0.01) ). Immunohistochemistry showed decreased IGF-IR expression. The effect of Trail on IGF-1R-siRNA A549 cell proliferation was decreased (P <0.05) and the apoptosis rate was significantly increased (P <0.01). Conclusion IGF-1R-siRNA expression vector has RNA interference. After the expression of IGF-1R decreased, the proliferation of A549 cells slowed down and the sensitivity to Trail-induced apoptosis increased.