Alteration In Mitochondria Of Lung-Bronchial Pithelial Cells And RNA Expression Upon Exposure To Radon And Alpha Particles

Objective：Radon is the second leading cause of lung cancer after smoking. Health hazard ofradon and its progeny to lung and bronchial epithelial cells come from high LETirradiation of its alpha particles. Mitochondria DNA （mtDNA） is one of the majortargets of carcinogens. This study is to investigate the adverse effects of radon gas andits alpha particles on lung tissues of rats as well as on human bronchial epithelial cellswith knockdown of mtDNA （ρ~-）, to observe alterations in mitochondrial structure andfunction during the cell transformation, and to screen differentially expressedmRNA/miRNA between the ρ~+and ρ~-cells upon alpha irradiation, in order to provideexperimental data for exploring potential mechanisms underlying malignanttransformation induced by radon.Methods：24healthy male Wistar rats were exposed to radon in a multi-function ecologicalradon room at a constant concentration to cumulative doses of100,200and400WLM（Working Level Month）. HBE cells were exposed to radon at20000Bq/m3for20minand repeated in3days to get first generation （Rn1, and continued to obtain Rn10andRn30.）.BEAS-2B cells were irradiated with α particles at doses of0.1,0.25,0.5and1.0Gy for2and4times respectively. A cell model with knockdown of mtDNA （ρ~-）was generated by the treatment of ethidium bromide （EB）. Real-time PCR was used todetect changes in mtDNA copy number, and the mitochondrial membrane potential,intracellular reactive oxygen species （ROS） and cell cycle were analyzed by flowcytometry. Cell transformation was reflected by resistance to serum, proliferationpotential, anchorage independent growth in soft agar and Transwell invasion test. The NimbleGen-135K mRNA chip and miRCURYTM chip were used to reveal changes inmRNA and miRNA expression, and Q-PCR was performed to further confirm the data.Results：Damages to the lung tissue of rats after radon inhalation include oxidative injury,increase in8-OHdG amount and mtDNA copy number, and decrease in TAOC level.Cell cycle related genes of P53, RB1, MDM2, MDM4, CDK2and CDK4weredown-regulated. The cell model of BEAS-2B with knockdown of mtDNA （ρ~-） andα-irradiation was established. Cells irradiated and then cultured for generationsevidenced malignant transformation by gaining anchorage independent growth andinvasion ability. Mitochondrial membrane potential, mtDNA copy number and ROSlevel were all elevated after α-irradiation. Numbers of the G1and G2phase in cell cycledeclined but the S phase increased. Among the cell cycle related genes, CDK2andCDK4were up-regulated, indicating an acceleration of the cell cycle from G1to Sphase. Up-regulation of P53, RB1, MDM2and MDM4also indicated an activation ofthese genes in regulation of cell cycle. The screening of mRNA expression revealedup-regulated genes functioning in cell inherence, apoptosis and cell differentiation, anddown-regulated genes functioning in protein translation, negative transcription factors,immunologic response and G protein signal. The up-regulated genes were involved inactivation of signal pathways of MAPK, adhesion molecular, amino acid metabolisminsulin metabolism and WNT, and in inhibition of TGF-β, purine metabolism, bladderand small cell lung cancer. In ρ~--P40cells, the up-regulated genes were functioning incell apoptosis, oxidative stress, protein phosphorylation, cell inherence and proteinsynthesis, and the down-regulated genes were functioning in cell inherence, intercellularsignal transduction, differentiation and hypoxia response. The up-regulated genes wereinvolved in activation of signal pathways of receptor interaction, MAPK, adhesionmolecular, amino acid metabolism, systemic lupus erythematosus, cell cycle, P53andsmall cell lung cancer, and in inhibition of TGF-β, axon forward, ubiquitin-mediatedprotein, neural active ligand and WNT. The pathway related to small cell lung cancerchanged in a way that it was inhibited in ρ~+cells but activated in ρ~-cells. In miRNA thatdifferentially expressed and related to mitochondria, let-7was up-regulated in ρ~+-P40 cells but down-regulated in ρ~--P40cells. A target gene prediction from the geneinteraction network indicated that COL12A1was target gene of miR-107in both theρ~+-P40and ρ~--P40cells. Target genes of FBN1and CUL1of miR-494wasup-regulatedup-regulated in ρ~+-P40cells but down-regulated in ρ~--P40cells, with itstarget genes of COL1A1, CDKN1A, CDK6and CLDN1up-regulated only in ρ~--P40cells, indicating that the knockdown of mtDNA could activate the function of thesetarget genes.Conclusions：1. An animal model of radon inhalation and an in vitro cell model of α irradiationwere established, and a ρ~-cell line with mtDNA knockdown was created by EBinduction. The toxic effects of radon/α irradiation on copy numbers and membranepotential of mitochondria, intracellular reactive oxygen species as well as celltransformation were determined to characterize the ρ~-cells.2. Upon α irradiation, some cell cycle related genes were up-regulated to acceleratethe cells from G1to S phase. This result was identical to that obtained from the in vitrotest of radon-exposed cells, confirming that the toxic effect of radon on cells was due tothe α-irradiation emitted by radon progeny.3. High-through chip screening revealed a set of differential expressed mRNAs andmiRNAs between ρ~+and ρ~-cells and between pre-and after α irradiation. Thedifferential expression of some cell cycle related genes such as CDK2andCDK4, aswell as some miRNA such as miR-494and let-7c in ρ~+and ρ~-cells suggested animportant role of mitochondria in cell cycle regulation and cell transformation.