hnRNP B_1对人肺癌A549细胞DNA-PK活性及细胞周期、凋亡的影响  

Effects of hnRNP B_1 on DNA-PK Activity, Cell Cycle and Apoptosis in Human Lung Adenocarcinoma Cell Line A549

作　　者：韩娟[1] 李为民[1] 唐凤鸣[1] 蒲丹[1] 陈文彬[1] 

机构地区：[1]四川大学华西医院呼吸内科,成都610041

出　　处：《四川大学学报（医学版）》2008年第5期815-818,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30270587);教育部博士点基金(批准号20050610056)资助

摘　　要：目的探讨核内不均一核糖核蛋白B1(heterogeneous nuclear ribonucleprotein B1,hnRNP B1)对肺癌细胞抑癌基因DNA依赖蛋白激酶(DNA-dependent protein kinase,DNA-PK)的活性、细胞周期和凋亡的影响。方法根据干扰hnRNP B1基因序列的两段不同靶序列构建2个hnRNP B1的siRNA抑制表达质粒A、D,转染人肺癌A549细胞。实验分组为重组质粒A、D转染的A549细胞、空质粒转染的A549细胞、未转染的A549细胞、预先用DNA-PK抑制剂NU7026(10μmol/L)作用1h的重组质粒A、D转染的A549细胞。Western blot检测各组细胞hnRNP B1表达。采用DNA-PK检测试剂盒检测DNA-PKcs激酶活性。以流式细胞仪检测细胞周期及凋亡。结果hnRNP B1 siRNA抑制肺癌细胞hnRNP B1表达;转染hnRNP B1 siRNA细胞的DNA-PK活性、凋亡率较未转染组明显增高(P均<0.05);G1期细胞明显增多(P<0.05),S期细胞减少(P<0.05)。预先用NU7026处理的转染hnRNP B1 siRNA细胞组与未处理的转染组比较,其G1期细胞明显降低(P<0.05),S期细胞明显增多(P<0.05),凋亡率明显降低(P<0.05);DNA-PK酶活性和细胞凋亡率成明显的正相关(相关系数r=0.817,P<0.01)。结论hnRNP B1可以使DNA-PK酶活性降低,从而影响细胞基因组的稳定及参与调控肺癌细胞周期与凋亡。Objective To explore the effects of hnRNP B1 on DNA-PK activity, cell cycle and apoptosis in human lung adenocarclnoma cell line A549. Methods 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 μmol/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. Results 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 ceils decreased ,that of S ceils increased and cell apoptosis were significantly inhibited. DNA-PK activity was significantly positive correlation with the rate of apoptosis. Conclusion hnRNP B1 could affect the stability of cell genome and regulate the cell cycle and apoptosis by inhibiting DNA-PK activity.

关 键 词：DNA依赖蛋白激酶 核内不均一核糖核蛋白B1 肺癌细胞A549 

分 类 号：R734.2[医药卫生—肿瘤] R73-37[医药卫生—临床医学]