Correlation Between RNA Oxidation And Senescence In C.Elegans,and The Possible Mechanism

Objective To discuss the correlation between RNA oxidation and lifespan in C.elegans.Content Conduct the enzymatic activity assay in vitro of C.elegans NDX-4protein. Build up a C.elegans model with increased RNA oxidation. Observe the change of lifespan in C.elegans with elevated RNA oxidation and disscuss its possible mechanism.Methods First we constructed NDX-4clone vector and induced its exipression in vitro, and detected its hydrolytic activity by HPLC-UV technology. Then, taking ndx-4deficient C.elegans RB1054together with exogenous substrate of different concentrations, we dectected their levels of RNA oxidation by LC-MS/MS technology, represented by the amount of8-oxoG (numbers in every106G) in RNA and built up a model with elevated RNA oxidation. Next, using the group treated with KHB as a control, we observed the change of lifespan in RB1054treated with exogenous substrate. Besides, we assayed the levels of RNA oxidation by LC-MS/MS in RB1054collected in different time0.5,1,2,4,12,16,20hours after exogenous stimulation and observed the metabolic law of oxidized bases in RB1054in this case. Finally, establish the let-363mRNA dectecting method by Real-time RT-PCR techinology, and use Real-time RT-PCR techinology to analyse the change of let-363expression level, which represented by the value of2-△△Ct as KHB a control, in RB1054treated with exogenous substrate in different time.Results Analysis of HPLC-UV showed that both8-oxodGTP and8-oxoGTP were converted into corresponding disphosphate-and monophosphate-form. We succeeded buiding up the model with increased RNA oxidation. The lifespan of RB1054with elevated RNA oxidation was shorter than the control group. As observed in the metabolic law of oxidized bases in RB1054with substrate stimulation:the amount of8-oxoG was most at4h and was nearly decreased to the control level at8h after8-oxoGTP incubation. Results of analysis by Real-time RT-PCR techinology showed that the let-363expression level tended to be changed with time after8-oxoGTP treatment. Conclusion NDX-4protein in C.elegans has the ability to hydrolyze8-oxodGTP and8-oxoGTP. Elevated RNA oxidation can cause lifespan shorter in C.elegans. Whether the change of let-363expression level caused by increasing RNA oxidation is associated with shorten lifespan needs the further experimental verification.