Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

AIMS AND BACKGROUND: Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. METHODS AND STUDY DESIGN: We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. RESULTS: We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. CONCLUSION: Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

[The inhibitory effects of siRNA expression vector on cell proliferation and expression of hnRNPB1 gene in lung cancer A549 cells].

OBJECTIVE: To investigate the inhibitory effects of specific siRNA on the expression of heterogeneous nuclear ribonucleoproteins (hnRNPB1) and cell proliferation in lung cancer A549 cells. METHODS: After the construction of siRNA expression vector for hnRNPB1, cultured A549 cells were tansfected with the specific siRNA vector, so RNA of A549 cells was interfered by RNA interference technique. The cells were retrieved at 1st, 4th and 6th week after transfection, cell cycles and cell apoptosis were measured with flow cytometry, the mRNAs levels of hnRNPB1, were detected by fluorescence quantity RT-PCR, the protein levels of hnRNPB1 was detected by Western blot technique. RESULTS: It was found that plasmid-derived siRNAs could inhibit hnRNPB1 mRNA and protein expression. The expressions of hnRNPB1 mRNA and protein were decreased about 46%-73%, and 77.69%-83.04% respectively. We also observed that siRNA in A549 cell suppressed the cell proliferation by increasing the amounts of G1 cells, and induced cell apoptosis. CONCLUSION: These results demonstrated the effectiveness of siRNA in lung caner cell, which indicated a possible clinical therapy method for lung cancer.

[RNA interference of hnRNP B1 gene in human A549 lung cells].

OBJECTIVE: To test the effect of RNA interference of hnRNP Bx gene in human lung cell line A549. METHODS: The RNAs of cell line A549 were interfered by the RNA interference technique. The hnRNP B1 mRNAs were detected by fluorescence quantity RT-PCR. The proteins of hnRNP B1 were detected by Western blot technique. The growth rate of the cells was analysed by MTT. RESULTS: The growth of the cells was suppressed significantly. The mRNA and protein expressions of hnRNP B1 were inhibited. CONCLUSION: The expression of hnRNP B1 can be inhibited by RNA interference. RNA interference could become a possible clinical therapy for lung cancers.

[Effects of hnRNP B, siRNA on the expression of p53 in human lung adenocarcinoma cell line A549].

OBJECTIVE: To explore the effects of hnRNP B1 on the expression of p53 in human lung adenocarcinoma cell line A549. METHODS: Two lines of A549 cells transfected by hnRNP B1 specific siRNA (recombinant plasmid A and D), which had a high transfection efficiency, were established as experimental groups. One line of A549 cells and one line of A549 cells transfected by pure plasmid served as controls. All of the cells were collected after treated with 10 micromol/L cisplatin for 24 h. The expressions of p53 mRNA and protein were detected by real-time RT-PCR and Western blot analysis. The expression of Phospho-P53 protein was detected by western blot analysis. RESULTS: No significant differences in the expression of p53 mRNA and protein among the groups were found. The expression ratio of Phospho-P53 protein in the cells transfected by hnRNP B1 siRNA were 0.87 +/- 0.06 and 0.75 +/- 0.06, which were significantly greater than those in the A549 cell (0.21 +/- 0. 04) and in the A549 cells transfected by pure plasmid (0.30 +/- 0.08) (P < 0.01). CONCLUSION: hnRNP B, specific siRNA can up regulate the activity of p53 in A549 cells.

[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. CONCLUSION: hnRNP B1 could affect the stability of cell genome and regulate the cell cycle and apoptosis by inhibiting DNA-PK activity.

[Expression of hnRNP K in lung adenocarcinoma cells].

OBJECTIVE: To examine the effects of hnRNP K on the growth and apoptosis of lung adencarcinoma cells in vitro. METHODS: The expressions of hnRNP K were detected by the SP method of immunohistochemistry in 70 lung adencarcinoma tissue samples. The hnRNP K siRNA was transfected into human lung adencarcinoma cell line A549 with Lipofectamine 2000. The cells transfected with siRNAn and untreated served as controls. The inhibitory effect of siRNA on the expression of hnRNP K mRNA and protein was detected by RT-PCR and Western blot. The change in cell cycling and cell apoptosis of siRNA-treated cells was detected by flow cytometry. RESULTS: Positive expressions of hnRNP K were found in 38.5%, 95.2% and 91.7% of lung adencarcinoma tissues in tumors with a diameter of < or =3 cm, 3-5 cm and > or =5 cm respectively (P < 0.01). The expressions of hnRNP K mRNA were significantly inhibited in siRNA- transfected cells compared with those in controls (P < 0.05). hnRNP K protein decreased in hnRNP K siRNA-transfected cells. siRNA targeting human hnRNP K inhibited the growth of lung adencarcinoma cell line A549 and the distribution of the cell cycle, with an apoptosis rate of 4.79%. Twenty four hours after transfection, the number of cells in G0/G1 phase increased from 37.21% to 85.60%, while the number of cells in S and G2/M phases decreased from 47.71% and 13.00% to 13.50% and 0.32%, respectively. CONCLUSION: The expression of hnRNP K is closely correlated with the size of lung adencarcinoma. hnRNP K siRNA inhibits the growth aNd increases the apoptosis of lung adencarcinoma cells.