High Throughput Screening Of Radiation-related Molecular And Validation

Ionizing radiation (IR) can induces a lot of genes and proteins expression and lead to cell/tissue damage. These IR-responsive genes and proteins are the basic for studying the molecular meachnisms of radiation damage, discovering new targets for radiation protection and biomarkers. Microarray and proteomics techniques have offered efficient platforms for high thoughput screening these genes and proteins. In this study, we used this two platforms to screen IR-responsive genes and proteins in cells and tissues, a candidate target for radiation protection will be discussed. All the results are as following:(1) In this section, the IR-related cellular responsive of AHH-1 was analyzed, then the whole human genome bioarrays were used to examine gene expression profiles in AHH-1 cells exposed to IR. The microarry analysis revealed a set of IR responsive genes, including 906 genes at 4 h and 789 genes at 24 h after exposure to 5 Gy IR. There were 115 genes changed in both time points, most of the IR-responsive genes are functionally related to cell cycles, apoptosis, and signal pathways. Twelve of the IR-responsive genes were validated by RT-PCR, the results were in agreement with those from microarrays.(2) In order to screen the new drug targets for radiation protection from the IR-responsive genes, 10 genes were screened and validated for their effects on radiation protection using antisense oligonucleotides. The results showed that down-regulation of Caspase-4 expression by antisense oligonucleotides significantly inhibited cell death induced by IR. The radiation survival curves derived from colony formation assays also showed that inhibition of Caspase-4 activity by AS significantly increased cells radio-resistance and protected cells from undergoing apoptosis induced by IR.. Taken together, the results suggested that Caspase-4 may is a candidate new target for radiation protection.(3) To identify proteins involved in the processes of cellular and molecular response to radiation damage in liver, C57BL/6 mice were irradiated for 20 Gy, 24 h later, subcellular proteins were purified and subcellular proteomics was used to identify differentially expressed proteins from mice liver nuclear,mitochondria and cytosol. A total of 14 proteins were identified as differentially expressed proteins in the nuclear compartment of the irradiated liver compared to control, 15 proteins were identified differentially expressed in mitochondria and 8 proteins were identified differentially expressed in cytosol. Most of these proteins are involved in oxidative stress injury, energy metabolism, lipid metabolism, amino acid and protein metabolism, molecular chaperon and inflammatory response. These proteins may play an important role in radiation-induced liver injury. Two of these proteins were validated by RT-PCR and Western blotting, the results were confirmatory of proteomic data.In conclusion, through microarray analysis, a panal of IR-responsive genes were identified, Caspase-4 plays an important role in radiation-induced apoptosis and is a potential target for radiation protection; a total of 37 differentially expressed proteins were identified from mouse liver nucleus, mitochondria and cytosol, which may provided clues for understanding the mechanism of injury in liver induced by irradiation and potential targets for therpy.