Study On The Changes And Interactions Of Mitochondrial Gene Cytochrome Oxidase Expressions Induced By Irradiation

ObjectiveThe mitochondria is cell organism of cell oxidative phosphorylation and mitochondrial DNA is the only extra-nuclear genetic material, which is coding37genes (including22tRNA genes,2rRNA genes and13polypeptide genes). Mitochondrion plays a very important role in cell oxidative phosphorylation (OXPHOS) and ATP synthesis, so that it provides energy for cellular activity.It is found that the copy number of mitochondrial genome is higher than that of nuclear genome, lower complexity, but more sensitive to ionizing radiation. The function of mitochondria is regulated by nuclear genome and the level of ATP is balanced by both of mitochondria genome and nuclear genome. We had found the expression of cytochrome oxidase (COX) genes is significantly up-regulated after radiation, which had provided the scientific basis for the study on the ionizing radiation-induced interaction between the nuclear genome and mitochondrial genome.This study aims to explore the radiation-induced gene expression changes of mitochondrial genes NRF-1, mtTFA, COX I, COX II, COX Ⅳ, COX Ⅴb; and then observe the gene expression changes after interference mtTFA expression. The cell activity and ATP concentration induced by ionizing radiation in human normal liver cell were also detected. So the present study can provide new data of molecular biological effects and scientific basis for screening sensitive markers for radiation damage. Methods1. The human lymphoblastoid cells AHH-1and normal liver cell line L02were irradiated with doses of0-15Gy60Co γ-rays at dose rate of1Gy/min.The gene expression alteration of COX I, COX II, NRF-1, mtTFA, COXIV, COX V were detected24h or48h after irradiation. Real-time PCR and Western blot were used to analyze the mRNA and protein level respectively. The cell activity and ATP level were also detected.2. In order to explore the interaction of mitochondrial gene and nuclear genes of COX, the mtTFA gene in L02was knocked down by RNA interference. Before and after knock-down the mtTFA gene, the gene expression alteration of COX I, COX II, NRF-1, mtTFA, COX IV, COXⅤ were detected24h or48h after irradiation. The cell activity and ATP level were also detected.Result:1. The dose-response relationship of COX I gene in AHH-1cells was established24h and48h after exposed to0-5Gy60Coγ-rays. The expression of COX II gene showed a good dose-effect relationship in the dose range of0-15Gy24h after irradiation. The expression of mtTFA gene showed a good dose-effect relationship in the range of0-15Gy24h and48h after irradiation, but other genes did not show good dose-response relationship.2. The expression of NRF-1, mtTFA, COX I, COX II, COXⅣ, COX V b genes on mRNA level in normal human liver cells L02showed a good dose-effect relationship24h and48h after irradiation in the dose range of0-15Gy60CoY-rays. The expression of mtTFA, COX I and COX Ⅱ genes showed a good dose-effect relationship in the dose range of0-10Gy24h after irradiation. The expression of COXIV and COXVb genes showed a good dose-effect relationship in the dose range of0-5Gy24h after irradiation. The expression of COX I gene showed a good dose-effect relationship in the range of0-8Gy48h after irradiation. COX I, COX II and mtTFA protein levels were significantly increased and showed a good dose-effect relationship24h after irradiated with60Coγ-rays. Although COX I, COX II and mtTFA genes expression levels were significantly increased48h after irradiated by60Coγ-rays, but all of them not showed a good dose-effect relationship.3. Before the knock-down the mtTFA gene, the expressions of NRF-1, mtTFA, COX I, COX II, COXIV, COXⅤb were increased comprehensively compared with the control group24h and48h after irradiation. After mtTFA knock-down, the expression of COX I, COX II were down-regulated, and the expression ofCOXⅣ, COXⅤb were significantly up-regulated, at the same time the expression of NRF-1did not change obviously.4. The concentration of ATP increased and cell activity decreased with increasing irradiation dose. After RNA interference the mtTFA, the concentration of ATP increased with the dose. Before the mtTFA knock-down, the concentration of ATP decreased with the irradiated doses.Conclusion:1. The expression of NRF-1, mtTFA, COX I, COX II, COXIV, COXⅤb genes on mRNA level in normal human liver cells L02were up-regulated24h and48h after irradiation. Six radiation sensitive mitochondrial genes have their own different optimal induction dose.2. The mtTFA gene regulates radiation-induced genes expression of COX I and COX Ⅱwhich coded by mitochondrial, but no regulates COXIV and COXⅤb genes coded by nuclear genome.3. The concentration of ATP in L02cells induced by ionizing radiation increased with the increased dose. That is the case after knock-down mtTFA gene, but the concentration of ATP decreased significantly. This might result from the COX I and COX II genes coded by mitochondrial.The present study demonstrates that ionizing radiation can up-regulate the expression of mitochondrial genes and nuclear genes. The model of knockdown mtTFA gene had successfully established which had down-regulated the expression of COX I, COX II genes. ATP also reduced to lead the cell to death. It can be proved that the mtTFA gene is up-stream gene of COX I, COX II in response to radiation. More important, mtTFA, COX I and COX II genes play an important role in the regulation of apoptosis pathways. The present study also provides the basic information for the research of radiation sensitivity.