The Regulation Of Human RNR Genes In Response Of UV Or Ag-nanoparticles-induced Stress Response

The ribonucleotide reductase (RNR) is the only crucial enzyme catalyzing the dNTP synthesis from de novo pathway in human cells, which is momentous for the regulation of dNTP pool. The stability of dNTP pool serves the foundation of cell proliferation and DNA repair. The precise regulation of dNTP pool also keeps the genome from labilization. Two aspects of work are revealed in this paper:The nucleotide excision repair (NER) is one of the most important manners of DNA repair. The XPC protein recognizing DNA damages also functions as a transcription regulator. To test whether this modulation can be done for RNR genes, we used the XP-C cell line (which XPC was mutated) and normal embryonic lung fibroblasts MRC-5cell line. After UVC treatment for these two kinds of cells, we tested the mRNA level and protein level of hRRM1, hRRM2and p53R2. We found that the transcription and expression of hRRM2was increased in XP-C cells, but it maintained stable in MRC-5cells. We also tested the transcription of E2F1and Rfx1, which possessing the potential regulation for hRRM2transcription, and regarded E2F1as the target for controlling the hRRM2transcription. Through performing ChIP experiment, we found that the XPC protein actually binds on the promoter of hRRM2gene in MRC-5cells, which represses the transcription of hRRM2in MRC-5cells under ther stress of UV treatment.The widely usage of silver nano-particles (AgNPs) in medical fields and commodities, which is along with drinking or intaking the caffeine in coffee or some medicines, will exerts a potential thread to human health. Firstly, I found the apt AgNPs and/or caffeine treating condition via HeLa cells. Then I chooesd MRC-5cells and H1299cells for my experiments. Through quantity-RT-PCR, I tested the transcriptional level of5pathways:1, p53and MDM2pathway;2, hRRM1, hRRM2and p53R2genes which belong to the RNR genes;3, E2F1, Bax and Apafl, which function in the apoptosis pathway;4, SOD1, SOD2and catalase, which are responsible for the elimination of ROS;5, p21and CyclinDl, which control the cell cycle progress. The results have shown that the transcription of p53, MDM2, Bax, Apaf1, SOD1, SOD2, catalase, p53R2and p21increased after added in caffeine to MRC-5cells. However, for the genes of RNR, the mRNA level of hRRMl didn’t change no matter whether the AgNPs and caffeine exist or not in these two kinds of cells. The transcription of hRRM2was apparently inhibited by caffeine. The results of Western Blot suggest that AgNPs can increased the expression of hRRM1and hRRM2in MRC-5and H1299cells, while caffeine can suppress the protein levels of these two proteins. To identiy the mechanism of this effect of inhibition, I introduced the inhibitor of proteasome, MG132to the treatment of the cells. In MRC-5and H1299cells, expression of hRRM1was still repressed by caffeine under the treatment of MG132. However, the expression of hRRM2was recoverd after MG132treatment in p53-nulled H1299cells, which I cannot find in MRC-5cells. This suggests that the p53protein may regulate the degradation of hRRM2to keep the dNTP pool stable.