Adverse Effect Of Radon On Mitochondria Of Human Bronchial Epithelial Cells And Changes In RNA Expression

Objective:Both Epidemiological investigation and experimental research confirmed the correlation between exposure of high level of radon and lung cancer. Bronchial epithelial cells, as well as their nuclei, are the targets of radon and its progeny. Mitochondrial has been shown to play an important role in the regulation of bystander effects of a particle, and mitochondrial DNA is one of the targets of carcinogens. This study is to set up a model of human bronchial epithelial cells (HBE) with knockdown of mitochondrial DNA (p") to observe the adverse effects of radon irradiation, and to screen differentially expressed mRNA/miRNA beteen p+HBE and p" HBE cells upon radon exposure, in order to provide experimental data for exploring potential mechanisms underlying malignant transformation induced by radon.Methods:p" HBE cells were generated by treatment of ethidium bromide (EB), and exposed to radon gas in a special inhalation chamber. The exposure concentration was set to20,000Bq/m3and lasted for20min each time and repeated in3days. Ten generations (Rn10) and30generations (Rn30) were exposed to radon, and the copy numbers of mitochondrial were determined by Real-time PCR, the number, morphology, membrane potential of mitochondrial and intracellular reactive oxygen species (ROS) were labeled by live cell probes. The alternation of apoptosis and cell cycle distribution were evaluated by Annexin V and PI staining. The NimbleGen-135K mRNA chip and miRCURYTM chip were used to reveal changes in mRNA and miRNA expression, and Q-PCR was performed to further confirm the data.Results:Thirty days after the EB induction, the number of mitochondrial in p-HBE cells decreased77%of that in p+HBE cells. Intracellular mitochondrial DNA was reduced significantly and mitochondrial membrane potential (MMP) was decreased. After removal of uridine for3days, lots of cells could not survive, suggesting that the p"HBE cells were successfully established. Upon exposure to radon, the proliferation of p+HBE cells was accelerated, with decreased clonogenic rate and survival fraction. In p’HBE cells, the clonogenic rate and survival fraction were increased, implicating a more severe damage by radon. The ROS level in both cells were elevated by radon exposure, but the level in p-HBE cells was only48%of that in p+HBE cells. The membrane potential in p"HBE cells was lowered as a result of mitochondrial damage. The apoptotic rate of the p-HBE cells was also found lower than in the p+HBE cells, although both cells showed increased apoptosis when exposed to radon, suggesting a correlation between mitochondrial function and cell proliferation. DNA partial depletion could inhibit apoptosis. Cells in S phase increased and those in G1and G2decreased after radon exposure, indicating a G1and G2/M phase arrest and prolonged cell cycle. The screening of mRNA expression revealed an up-regulation of TFF1, TFF2and IGHG1, and down-regulation of NCOA7, FN1, EGR1, IL-8, RASEF and BNDF. The GO analysis showed that these mRNA mainly affect signal transduction and protein/enzyme activity. In radon exposed p+HBE cells, BICC1, ASNA, KIAA1045were up-regulated, and GNAB, TSGA2, BAX, PKP2and GNPDA1were down-regulated. In radon exposed p-HBE cells, SPRR2E, SPRR2A, and SPRR2D were up-regulated and HAND2and FCRLM1were down-regulated, with functions in receptor signal, signal transduction and protein synthesis. The screening of miRNA expression revealed an up-regulation of miR-107, miR-3651and miR-3074-3p, and a down-regulation of miR-1227and miR486-5p in radon exposed p+HBE cells. In radon exposed p-HBE cells, miR-1919-5p,miR-3157-3p and miR-484were up-regulated, and miR-3607-3p, miR-98and miR-1275were down-regulated.Conclusions:1. A p-HBE cell model was successfully established by EB induction. The copy numbers and membrane potential of mitochondria, intracellular reactive oxygen species, apoptosis and cell cycle were determined to characterize the p-HBE cells.2. Upon radon exposure, indexes reflecting cell growth, ROS production, membrane potential of mitochondria, apoptosis and cell cycle were altered indicating a correlation between mitochondrial disfunction and the cell damage.3. High-through chip screening revealed a set of differential expressed mRNAs and miRNAs between p+HBE and p-HBE cells and between pre-and after radon exposure. Bioinformatic analysis will help elucidate roles that nuclear DNA and mtDNA may play in signal transduction of radon induced lung cancer.