| Objective: The aim of the present study was to explore the role and mechanisms of p53 in mediation of energy metabolism in malignant transformation of human bronchial epithelial(BEAS-2B) cells induced by radon.Methods:(1) To establish the p53 knockout BEAS-2B cell model(p53 KO) by TALEN technology.(2) To establish the malignant transformed BEAS-2B and p53 KO cell models induced by radon. The BEAS-2B cells and p53 KO cells were exposed to radon for 20 min at a concentration of 20000 Bq/m3, and cultured for 3 days before the next exposure at the same concentration and duration. This procedure was repeated for 20 times until the cells turned to malignant transformation. Changes in cell proliferation, soft agar colony formation, cell migration rate and invasion ability were measured during the process.(3) Glutamine concentration, lactic acid and lactate dehydrogenase(LDH) were detected by biochemical techniques. The ATP level was determined by luminometer. Cell oxygen consumption rate(OCR) was detected by extracellular flux cytometry. Membrane potential(MMP) was measured by JC-1 fluorescence with flow cytometry. Mitochondrial copy number was analyzed by real-time PCR. The expression of p53, SCO2, TIGAR, IDH3 A, SDHA, HK-II, PFKM and PKM2 were determined with western blot.Results:(1) The gene sequencing result and p53 protein expression indicated that the p53 gene was knockout.(2) The number of soft agar colony formation and the invasion and migration rate in BEAS-2B-Rn and p53 KO-Rn cells were higher than that in BEAS-2B and p53 KO-Rn cells, with a shorter doubling time. Malignantl transformation occurred in BEAS-2B-Rn cells and p53 KO-Rn cells at 20 passages and 15 passages respectively.(3) In the process of cell malignant transformation induced by radon, the levels of glucose consumption, lactate, LDH and ATP significantly increased, while the oxygen consumption rate decreased. The mtDNA copy numbers in BEAS-2B-Rn cells increased from the 10 th passage, and continued to increasein later passages. On the other hand, the mt DNA copy numbers in p53 KO-Rn cells were lower than in the control and BEAS-2B-Rn cells. The MMP in transformed BEAS-2B-Rn and p53 KO-Rn cells significantly decreased in comparison with the passage control group.(4) The expression of p53 in BEAS-2B-Rn cells decreased after 15 th passage. The expression of SCO2 and TIGAR in BEAS-2B-Rn cells decreased from 20 th passage, and was lower in p53 KO cells than in BEAS-2B cells. The expression of SCO2 in p53 KO-Rn cells increased from 5th passage, and decreased after 20 th passage. No significant change of TIGAR expression in p53 KO-Rn cells after radon exposure.(5) The expression of IDH3 A and SDHA in BEAS-2B-Rn cells decreased from the 20 th passage. The expression of SDHA decreased when the p53 gene was knockout, with no obvious changes in IDH3 A expression. The expression of HK-II in BEAS-2B-Rn cells was higer than in BEAS-2B cells from 5 to 25 passages. The expression of PFKM and PKM2 in BEAS-2B-Rn cells increased from passages of 20 and 15 respectively. The expression of PFKM in p53 KO cells was higher than that in BEAS-2B cells.Coclusion:(1) A cell model of p53 gene knockout human bronchial epithelial was successfully established.(2) A cell model of malignant transformation induced by radon was successfully established.(3) The established models were applied to study the molecular changes in energy metabolism of transformed human bronchial epithelial cells. The results indicated that mitochondrial damage, altered energy metabolism pathway and p53 loss of function were combined to promote the malignant transformation of human bronchial epithelial cells induced by radon. Dysfunction of p53 in regulation of mitochondrial energy metabolism played an important role in malignant transformation induced by radon. |
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