The Effects Of Ionizing Radiation On DNA Damage Repair Protein DNA-PKcs And Its Binding RNAs

Objective:The most serious damage to human genomic DNA caused by ionizing radiation in the environment is DNA double strand breaks?DSBs?.When DSBs occur,the most important repair pathway of human cells is DNA-dependent Protein Kinase Catalytic Subunit protein?DNA-PKcs?is a core factor-led DNA nonhomologous end joining?NHEJ?.In recent years,a great deal of research has been conducted on DNA-PKcs,and DNA-PKcs as an upstream key protein kinase plays an important role in the NHEJ pathway.Therefore,we pay attention to the changes of DNA-PKcs protein kinase activity induced by ionizing radiation-induced gene double-strand break in human cells,and study the enhancement of kinase activity with radiation time dose effect,which can be used as a target protein for the study of human cell gene radiation damage repair mechanism.With the continuous research in recent years that DNA-PKcs protein can function by binding RNA,we further study the role of DNA-PKcs protein in the repair process of ionizing radiation damage from the perspective of RNA-protein complex.Methods:?1?The cell DSB model was constructed by using ⁶⁰Co?-ray irradiation in our hospital at room temperature.U2OS cells were irradiated with 8 Gy dose,and the cells were irradiated with no control group?con?and after irradiation?0.5,1,2,4,8 h?.The DNA-PKcs protein was observed by Western Blots at different time points after ionizing radiation.Phosphorylation of Ser2056 locus was followed by different doses?0,2,4,8 Gy?⁶⁰Co?-ray irradiation,and cells were collected at 2h,and DNA-PKcs protein and its Ser2056 locus were observed by Western Blots.Phosphorylation expression.At the same time,in order to exclude cell-specific experiments,different doses?0,2,4,8 Gy?of ⁶⁰Co?-rays were used to irradiate HeLa cells,293T cells and HepG2 cells,and Western Blots were used to observe the DNA-PKcs protein Ser2056 in each cell at 2h after irradiation.Point phosphorylation expression.?2?Using DNA-PKcs as the target protein,U2OS cells were irradiated with8Gy ⁶⁰Co?-rays,and the nucleoproteins of the samples were not irradiated?con?and after irradiation?0.5,1,2,4,8 h?.The specific RNA binding protein immunoprecipitation was carried out,and the protein and RNA in the precipitate were separated and extracted.Western Blots experiments were carried out to verify the enrichment effect of the antibody protein.The RNA quality was experimentally verified by RNA spectrophotometer and electrophoresis.High-throughput sequencing analysis?Nuohe Zhiyuan Company?experimental alternative RNA samples,for sequencing data results,using Fast QC for quality control and statistics,comparison with reference genes by BWA software,splicing alignment results using cufflink software And calculate the amount of expression.The difference between the control group?con?and the experimental irradiation group?0.5h,1h?differentially expressed genes was analyzed by differential analysis as the target genome for DNA-PKcs protein-binding RNA.Then functional annotations?GO annotations and KEGG annotations?were performed on the target genome,and the RNA molecules with higher enrichment were selected as candidate RNAs for subsequent studies and verified by qRT-PCR experiments.RNAs with large differences were selected as the final study objects.Functional experiments were performed using a method in which siRNA inhibits expression of a target RNA,and an experimental group which knocks down DNA-PKcs expression and an inhibitor of DNA-PKcs kinase activity was used for comparison.Results:?1?The enhanced DNA-PKcs protein kinase activity after ⁶⁰Co?-ray irradiation has a time effect.?2?The increase of DNA-PKcs protein kinase activity after⁶⁰Co?-ray irradiation has a dose effect.?3?The dose effect of ⁶⁰Co?-ray irradiation on DNA-PKcs protein kinase activity is not cell specific.?4?The selected DNA-PKcs antibody can effectively immunoprecipitate DNA-PKcs protein.?5?The RNA separation spectrophotometer obtained by experimental separation and purification has an OD260/280 range of 1.8-2.2,and the gel electrophoresis quality test RNA has a relatively complete and clear RNA band.?6?Three metabolic pathways with high enrichment were selected by KEGG pathway analysis and GO enrichment analysis.The five genes of the three enrichment pathways were ITGA3,ITGA5,ITGAV,CD44 and SDC4.?7?The expression of genes in qRT-PCR showed an increasing trend,and the expression differences of ITGA5 and SDC4 genes were the most obvious.?8?The expression of ITGA5 and SDC4 genes was inhibited by siRNA.The cell migration rate was significantly decreased in the experiment.The experimental results were consistent with the trend of lowering the cell migration rate by knocking down DNA-PKcs expression or using DNA-PKcs kinase activity inhibitor.Conclusions:?1?The expression of DNA-PKcs protein kinase activity in human cells has the time and dose effects of ionizing radiation.Therefore,DNA-PKcs protein can be used as a target protein for the study of radiation damage repair mechanism of human cell genes.?2?Successfully obtained a high-throughput RNA library for RNA binding to the U2OS cell DNA-PKcs protein complex.?3?DNA-PKcs protein and ITGA5,SDC4 genes may interact in cell motility.