Optimization Of Detection Methods For RNA Oxidative Damage And Analyze Cell RNA Oxidative Damage Under Different Conditions

Objective: RNA is a kind of biological macromolecules distributed in the cells widely,RNA molecules play many essential roles in cells, especially in the regulation of geneexpression and protein translation. RNA damage exist in physiological and pathologicalconditions, but it has not caused enough attention. Recently, oxidative RNA damage hasbeen reported in several neurodegenerative diseases including Alzheimer disease,Parkinson disease, etc. Therefore, the research of oxidative RNA damage will contributeto the understanding of disease mechanisms, and further provides the theoretical basisand methods for the diagnosis and treatment of related diseases. This study is to developand optimize the detection methods for RNA oxidative damage and analyze cell RNAoxidative damage of the different cells and tissues under different conditions, andexplore the connection between ataxia-telangiectasia mutated (ATM) protein kinaseactivation and RNA oxidative damage. Methods: First, reverse transcription blockingcombining with Double-Primer PCR: To establish both cell-free systems and cellsystems of RNA oxidative damage model using hydrogen peroxide (H2O2) and extracttotal RNA then reverse transcribe into cDNA. According to the target sequence ofmRNA5’ and3’ ends, two pair of primers were designed respectively. The real-timequantitative PCR were performed using primers which pair with the5’ end or3’end ofspecific mRNA respectively. We choose the host gene GAPDH and specially expressinggene p53to be determined, supposing that the more damages occur, the less integratedcDNA yield, namely the less cDNA which containing mRNA5’ end were transcripted.Second, develop competitive enzyme-linked immuno sorbent assay(ELISA): To coat the ELISA plates with8-OHG, which the most common oxidative modication to RNA, andincubate with RNA samples to compete binding to the special antibody15A3, then readOD450to determine RNA oxidative damage level, the more8-OHG in RNA sample, thelower OD450. Using the competitive ELISA, we determined RNA damage sensitivenessin both ATM mild and mutant cells, also ATM autophosphorylation at Ser1981inHEK293was detected by Western blotting (WB) after been transfected with oxidativeRNA. Results: In both cell-free systems and cell systems, the GAPDH and p53mRNAoxidative damage level treated by H2O2were significantly higher than control, and withthe increasing of H2O2concentration, mRNA oxidative damage were more severe.Furthermore we found that the damage detection sensitivity increased with reducingreverse transcriptase. We also demonstrated that RNA oxidative damage existed duringthe process of cell senescence and in tissues of the liver cancer; RNA oxidative damagelevel and ionizing radiation dose are in positive correlation. ATM cells is sensitive tooxidative stress than the wild-type cells, ATM is activated when oxidated RNAs weretransfected into the cells. Conclusion: Two detection methods for RNA damage havebeen developed and optimized in this study, which will provide technical support for thediagnosis and analysis of relative disease. On the other side, we proved that ATMactivation maybe involved in RNA damage response, and further research is needed toreveal the exact mechanism.