| Part1 Establish the mitochondrial DNA depleted cell line mode of non-small cell lung cancer H1299 and the changes of radiobiologyObjective Radiotherapy is one of the mainly treatment of NSCLC, but radioresistance can lead to failure of radiotherapy. At present, the relationship between mitochondrial DNA (mtDNA) and radiosensitivity is still conflictable. We are aimed to establish the mtDNA depleted H1299 cell line (ρ0 ells), and to investigate the effects of mtDNA depletion on radiosensitivity.Methods ρ0 cells were depleted of mitochondrial DNA by culturing chronically in the presence of low concentrations of ethidium bromide (EB), and then verified by culturing in medium without sodium pyruvate and uridine and by PCR amplification of total DNA using primer pairs specific for mitochondrial and nuclear DNA. Radiosensitivity was analyzed by cologenic formation assay. ATP test kits were used to measure intracellular ATP levels. DCFH-DA was used to detect intracellular reactive oxygen species (ROS) levels. Cell apoptosis and cell cycle distribution were measured by flow optometry. Cell cycle-related proteins ATM/ATR/CCNB1 were analyzed by Western blotting. Confocal fluorescence microscopy was used to detect yH2AX focis.Results We successfully established H1299ρ0 cell. Cologenic formation assay showed the H1299ρ0 cells were more radioresistance than ρ+ cells, the SF2 was 0.788±0.058, 0.525±0.072, respectively(P<0.05); Do was 2.993±0.028,2.119±0.012, respectively(P<0.05); Dq was 3.601±0.015,0.983±0.033, respectively(P<0.05). The ATP concentration in non-radiation group of H1299ρ0 and ρ+cells was 54.108± 11.652 umol/L,87.428±7.222 umol/L, respectively(P<0.05). After 6h post-radiation, the ATP concentration was 17.964±5.225umol/L,28.240±6.313umol/L, respectively(P<0.05).The ATP production of ρ0 cells was lower than ρ+ cells both with or without radiation. The ROS fluorescence intensity was 49.953±3.86,54.509± 5.35, respectively(P<0.05) in non-radiation group of H1299ρ0 and ρ+ cells. Both H1299ρ0 and ρ+ cells were increased in ROS levels after 30min post-radiation, the fluorescence intensity radios was 66.174±6.26,84.579±9.47, respectively(P<0.05). Both ρ0 and ρ+ cells all had higher ROS levels after irradiation, however, the increased ROS levels in ρ0 ells were significantly lower than ρ+cells. Furthermore, after 6Gy X-ray irradiation, ρ0 cells showed prolonged G2 arrest which compared to ρ+ cells. The G2/M phase arrest peak time was 24h and 12h, respectively. The G2/M phase cell distribution rate was 37.416±2.105,48.100±3.6915, respectively(P<0.05). Meanwhile, the expression levels of cell cycle related protein ATM/ATR/CCNB1 in ρ0 cells were significantly higher than ρ+cells(P<0.05). There’s no significant difference of spontaneous apoptosis between H1299ρ0 and ρ+cells, the apoptosis rates was 2.35±0.378,3.175±0.512,respectively(P> 0.05). However, the levels of radiation-induced apoptosis were increased both in H1299ρ0 and ρ+ cells. The radiation-induced apoptosis in ρ0 and ρ+cells was 5.716±1.183,12.75±2.523, respectively(P<0.05). The spontaneous DSBs in H1299ρ0 nd ρ+ cells was 10±5.215, 11±3.741,respectively(P>0.05).The γH2AX focis in ρ0 ells(18±2.408) were lesser than ρ+ cells(24±5.639)(P<0.05) after 30min of 2Gy radiation, which indicating the accelerated DNA damage repair of ρ0 cells.Conclusion H1299ρ0 cells were depleted of mtDNA could be established by culturing in the presence of low concentrations of EB. H1299ρ0 cells have showed decreased radio-sensitivity, reduced ATP and ROS levels, prolonged G2/M cell cycle arrest, anti-apoptosis and strong ability of DNA damage repair.Part Ⅱ Identification and bioinformatic analysis of differentially expressed genes in H1299ρ0 and ρ+ cellsObjective The first part of the study had showed that the radiosensitivity of H1299ρ0 cell reduced, but the exact mechanism is unclear. The present study is aimed to identifiy differentially expressed genes in mitochondrial retrograde signaling (RTG) pathways between ρ0 and ρ+ cells by using human microarray and analyze by bioinformatics.Methods H1299ρ0 and ρ+ were used as tool cells in the present study. Total RNA was prepared from ρ0 and ρ+ cells. After quality identification and fluorescent labeling, RNA samples were hybridized with the Agilent human genome expression microarray (the chip summary current understanding of the human whole gene information, representing more than 41,000 human genes and transcripts). mRNAs expression profiles were obtained. Raw data were normalized using Gene Spring Software 11.0 software and Quantile algorithm. KEGG database were used for GO and Pathway annotations on differentially expressed genes, respectively.Results A total of 2659 differentially expressd geness were identified (fold change≥3, P<0.05), including 1499 up-regulated and 1160 down-regulated genes between H1299ρ0 and p+ cells.These differentially expressed genes were mainly involved in cell adhesion, cellular process and regulation, material transport, stimulation and immune response, cell growth and death. GO analysis demonstrated that the down-regulated genes were mainly act as one part of the mitochondria function, Such as ATP synthesis, electron transfer, amino acid metabolism, TCA cycle and ribonucleoprotein complex. However, the genes that mainly responsible for DNA repair, the expression of mitochondrial proteins and transport regulation were up-regulated, such as cytoplasmic ribosomes, mitochondrial surface protein folding and DNA damage repair gene expression. Pathway analysis showed that the pathways in cancer(human), adhesion, calcium signal pathway, receptor ligand pathway, PPAR signaling pathway, metabolism, MAPK signaling pathway, cytokines, etc were significant diffenence. Real-time PCR was used to verify significant difference genes of interested, the mRNA levels of AKT2, mTOR, IKKs and CCNB1 were increased, while DBT and BAX were decreased. These results were consistent with microarray.Conclusion Differentially expressed genes identified in the present study may be involved in the regulation of radiosensitivity. Bioinformatics analysis demonstrated that the down-regulated genes were mainly act as one part of the mitochondria function and the up-regulated genes were responsible for DNA repair, the expression of mitochondrial proteins and transport regulation.The mRNA levels of AKT2, mTOR, IKKs and CCNB1 were increased, while DBT and BAX were decreased. The results indicating that the dysfunction mitochondrial may regulate radioresistance through RTG pathway of NF-KB/PI3K/AKT2/mTOR.Part Ⅲ The mechanisms of RTG pathway in H1299ρ0 cell that affecting radiosensitivityObjective To investigate the relationship between significant differences genes of RTG pathway in H1299ρ0 cells and radiosensitivity, and explore the possible intervention targets of radiosensitization.Methods H1299ρ0 and p+cells were used as objects of the present study. Western bloting was used to detect the protein expression levels of RTG pathway in NF-KB /PI3K/AKT2/mTOR, when combined ionizing radiation with or without AKT inhibitors.Results The phosphorylated protein levels in NF-KB/PI3K/AKT2/mTOR signaling pathway were increased after 6Gy ionizing radiation in H1299ρ0 cells, meanwhile the upstream/downstream proteins of HIF-la and Bcl-2/BAX were also increased. While 6Gy ionizing radiation combined with AKT2 inhibitor MK-2206 showed that the phosphorylated protein levels in NF-KB/PI3K/AKT2/mTOR signaling pathway were decreased, while the downstream apoptosis factor pro-Caspase3 increased.Conclusion The increased expression of RTG pathway NF-KB/HIF-1α/PI3K/AKT2/mTOR after mtDNA deletions was closely related to radioresistance, and AKT2 may be the intervention targets of radiosensitization. |
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