Nucleic Acid Oxidation Inhibition Of Gene Nudt5 In Cell Cycle Regulation And Aging

Mammalian cells are continuously exposed to ROS, which are produced by various types of electron transport reactions occurring during physiological metabolism. RNA is largely single-stranded, and thus, is more vulnerable to oxidative attacks than is DNA, in which the bases are protected by hydrogen bonding. Moreover, RNA is mostly distributed in the vicinity of mitochondria, which are the primary sites for ROS production. It seems, therefore, that RNA can be oxidized more easily than DNA. By now MTH1, NUDT5, PNP and YB1 have been reported to participate in the inhibition of RNA oxidation. MTH1 and NUDT5 hydrolyze the oxidized nucleoside diphosphates and/or triphosphates to the monophosphates avoiding incorporation of the oxidized nucleotide into RNA. For oxidatively damaged nucleotides that already exist in RNA, PNP and YB1 can specifically bind to these kinds of RNA. In this study, RNAi technic was used to investigate the biological function of thesed genes in vivo.To select the most efficiency siRNA sequences for MTH1, NUDT5, PNP and YB1, three sequences for each were designed and the most efficiency siRNA was selected by RT-qPCR. Plasmid containing shNUDT5 was constructed and transfected in to HeLa cells to select NUDT5 stable knockdown cells. With LC-MS/MS, no significant oxidation of DNA and RNA in shNUDT5 cells was observed comparing with the control shGFP. Significant reduction of cell viability and G1/S delay was detected while no apoptosis was induced in shNUDT5 cells. Key proteins for preventing the G1-S transition, such as p53, p16 and Rb were increased, while the phosphorylation of Rb was decreased. To determine whether such a delay in cell cycle progression may be induced as a result of RNA oxidation, we examined the effects of 8-oxoGTP introduction to cells by hypotonic shock. By the direct input of 8-oxoGTP into HeLa cells, the amount of 8-oxoG-containing RNA increased approximately two-fold compared to that of normal cells. Under these conditions, however, no delay in the G1 phase were observed.To investigate the role of NUDT5 in senescence, retrovirus particles containing shNUDT5 sequence were produced with HEK293T cells and transfected into human primary cells IMR-90. The results showed that cell viability was significantly decreased in NUDT5 knockdown cells. Significant increase of senescence associated P-gal was observed in shNUDT5 primary cells compared with the control cells. Significant increase in apoptosis and decrease in population doubling were induced after NUDT5 was knocked down. siRNA may be of off-target, in order to rescue the phenomenon induced by NUDT5 knockdown, three site mutations were introduced into the target sequence of siRNA in NUDT5 expreesion plasmid. The site mutations were all non-sense, so the amino acid sequence of NUDT5 protein was not changed. The mutated mRNA of NUDT5 cannot be recognized by the original siRNA. The effect of this rescue experiment was confirmed in HeLa cells.The klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. Klotho can protect cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. In this study we investigated the exprrssion pattern of NUDT5 and amount of 8-oxoG in DNA and RNA in the hippocampus of Klotho-Knockout mouse. Our results showed that there was no increase of oxidation in both DNA and RNA, NUDT5 expression pattern was also not changed in klotho knockout mouse.