Han Juan, Li Wei-min, Tang Feng-ming, Pu Dan, Chen Wen-bin
Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Sep;39(5):815-8.
To explore the effects of hnRNP B1 on DNA-PK activity, cell cycle and apoptosis in human lung adenocarcinoma cell line A549.
hnRNP B1 siRNA expression vectors (recombinant plasmid A and D) were constructed according to the different targeting sequences of hnRNP B1 gene. The recombinant eukaryotic expression plasmid A and D were identified by PCR and sequence analysis, and then were transfered into A549 cells respectively by Lipofectamine 2000, with or without preincubation of 10 micromol/L NU7026 (a specific inhibitor of DNA-PK) for 1 h. The expression of hnRNP B1 was measured by Western blot. DNA-PK activity was detected with SigmaTECT DNA-Dependent Protein Kinase Assay System. Cell cycle and apoptosis were analyzed by flow cytometry.
The expression vectors were successfully constructed. The expression of hnRNP B1 protein were reduced in the cells transfected with hnRNP B1 siRNA. The activity of DNA-PK in A549 cells transfected by hnRNP B1 siRNA was significantly higher than that of untransfected cells (P < 0.05). After the transfection of hnRNP B1 siRNA, the cells in G1 phase increased but those in S phase decreased, while the rate of apoptosis increased. With the treatment of NU7026, the number of G1 cells decreased,that of S cells increased and cell apoptosis were significantly inhibited. DNA-PK activity was significantly positive correlation with the rate of apoptosis.
hnRNP B1 could affect the stability of cell genome and regulate the cell cycle and apoptosis by inhibiting DNA-PK activity.
探讨异质性核糖核蛋白B1（hnRNP B1）对人肺腺癌细胞系A549中DNA依赖蛋白激酶（DNA-PK）活性、细胞周期及细胞凋亡的影响。
根据hnRNP B1基因不同的靶向序列构建hnRNP B1小干扰RNA（siRNA）表达载体（重组质粒A和D）。通过聚合酶链反应（PCR）及序列分析对重组真核表达质粒A和D进行鉴定，然后分别用脂质体2000转染A549细胞，转染前或转染时用10 μmol/L NU7026（DNA-PK特异性抑制剂）预处理1小时。采用蛋白质免疫印迹法检测hnRNP B1的表达。用SigmaTECT DNA依赖蛋白激酶检测系统检测DNA-PK活性。通过流式细胞术分析细胞周期及细胞凋亡情况。
成功构建表达载体。转染hnRNP B1 siRNA的细胞中hnRNP B1蛋白表达降低。转染hnRNP B1 siRNA的A549细胞中DNA-PK活性显著高于未转染细胞（P<0.05）。转染hnRNP B1 siRNA后，G1期细胞增多，S期细胞减少，同时细胞凋亡率增加。经NU7026处理后，G1期细胞数量减少，S期细胞数量增加，细胞凋亡受到显著抑制。DNA-PK活性与细胞凋亡率呈显著正相关。
hnRNP B1可通过抑制DNA-PK活性影响细胞基因组稳定性，调控细胞周期及细胞凋亡。
