摘要目的 研究亲环素A(CyPA)基因在非小细胞肺癌中的功能.方法 设计并合成4对针对CyPA siRNA靶序列的寡核苷酸单链,退火形成双链DNA,与经Hpal和Xhol酶切后的pGCL-GFP载体连接产生Lv-shCyPA慢病毒载体,该载体含U6启动子和绿色荧光蛋白(GFP),经聚合酶链反应(PCR)筛选阳性克隆并测序鉴定.将Lv-shCyPA、pHelper1.0和pHelper2.0质粒共感染包装细胞293T细胞,包装成假病毒颗粒并感染非小细胞肺癌A549细胞,经免疫印迹法(Western blot)检测筛选CyPAsiRNA有效靶点和慢病毒颗粒,采用流式细胞技术分析细胞周期和凋亡率,进行裸鼠移植瘤实验.结果 PCR和测序结果证实,Lv-shCyPA慢病毒载体构建正确,经Western blot法筛选最有效的CyPA siRNA病毒颗粒滴度为2×10 9TU/ml,感染A549细胞凋亡率(5.01%±0.57%)增加,细胞周期G2-M期比例(11.40%±0.68%)减少,裸鼠移植瘤瘤体生长明显减缓,与未感染病毒的A549细胞细胞凋亡率(0.35%±0.17%)及G2-M期比例(14.52%±1.19%)相比,差异均有统计学意义(P＜0.05).结论 CyPA基因沉默在非小细胞肺癌的发生中有一定的抑瘤作用,对非小细胞肺癌的预防和分子流行学研究提供了新的理论依据.

abstractsObjective To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.Methods First,four target sequences were selected according to CyPA mRNA sequence.The complementary DNA containing both sense and antisense oligonucleotides were designed,synthesized and cloned into the pGCL-GFP vector,which contained U6 promoter and green fluorescent protein (GFP).The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA,and it was confirmed by PCR and sequencing.Next,it was cotransfected by Lipofectamine2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles.At the same time,the packed virus infected non-small cell lung cancer cell (A549),the level of CyPA protein on 5 d after infection was detected by Western Blot to screen the target of CyPA.A549 was infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice.Cell cycle and apoptosis were measured by FCM.Results It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully.The titer of concentrated virus was 1×10 7 TU/ml.Flow cytometric analysis demonstrated G2-M phase (11.40%±0.68% )was decreased relatively in A549/Lv-shCyPA compared with control groups (14.52%±1.19%)(P＜0.05).The apoptosis rate of A549/Lv-shCyPA (5.01%±0.5%)was higher than control groups (0.35%±0.17%)P＜0.05).Visible tumors were only detectable on 6th day after inoculated by A549/Lv-shCyPA.The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size on 38th days after inoculation compared with the control group(P＜0.05).Conclusion Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA,induces the NSCLC cell apoptosis,and inhibits the tumor growth.Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.