[Effects of hnRNP B1 on DNA-PK activity, cell cycle and apoptosis in human lung adenocarcinoma cell line A549].
OBJECTIVE
To explore the effects of hnRNP B1 on DNA-PK activity, cell cycle and apoptosis in human lung adenocarcinoma 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 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.


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 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.


CONCLUSIONS
hnRNP B1 could affect the stability of cell genome and regulate the cell cycle and apoptosis by inhibiting DNA-PK activity.
Han
Juan
J
Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, China.
Li
Wei-min
WM
Tang
Feng-ming
FM
Pu
Dan
D
Chen
Wen-bin
WB
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C439768:hnRNP A2; D051747:DNA-Activated Protein Kinase