The Role Of Hsp90 In MRE11-RAD50-NBS1 Complex Related DNA Double Strands Break Repair Signaling Pathway In Oxidative Stress

BackgroundEnvironmental stimuli such as ionizing radiation,heat shock,hypoxia,environmental toxicants,ultraviolet radiation induce oxidative stress.Cell respond to DNA double strands break by activating complex DNA damage repair responses.DNA damage responses include recognition of DNA damage signaling pathways,recruitment of DNA repair factors,activation of DNA repair pathways,cell cycle arrest and apoptosis.DNA double strands break are repaired mainly by homologous recombination(HR)and non-homologous end-joining(NHEJ).DNA double strands break repair depends on a series of key protein molecules recognizing the site of injury,binding,activating the downstream pathway cascade reaction.Whether proteins with conformational aberrations can be re-folded correctly or degraded in time are critical to the maintenance of normal function and survival of cells under oxidative stress.In the protein quality control system,the molecular chaperone Hsp90 and its co-chaperone molecules help the substrate protein refolding and degradation plays a very important role.In the DNA damage repair pathway,MRN complex have been identified as the Hsp90 substrate protein.MRN protein complex by MRE11,NBS1,RAD50 three proteins assembled.Homologous recombination(HR)and non-homologous end joining(NHEJ)were used to identify the site of DNA damage by MRN complex,which is the initiation factor of DNA double strand breaks repair pathway.When the Hsp90 molecular chaperone function defects,will lead to substrate protein of DNA damage repair instability and function depression.It is reported that MRN protein complex is the substrate of Hsp90.So whether Hsp90 can affect the DNA double strands break repair by regulating the activity of MRN complex under oxidative stress.Objective1.To establish model of oxidative stress and evaluate the effect of oxidative stress and the biological effect of DNA double strands break damage.2.To explore the relationship between Hsp90?,MRN protein complex and y-H2AX under oxidative stress and its mechanism.Methods1.Cell Counting Kit-8 was used to detect cell viability.2.The cell cycle were measured by flow cytometry.3.DNA double strands break were detected by comet assay4.Western Blot was used to detect the expression of apoptotic protein(PARP and caspase-3)??-H2AX and MRN complexes.5.PCR to detect the mRNA level of the MRN complex.6.Immunofluorescence to detect the focus of y-H2AX and the formation of MRN complexes and y-H2AX.Results1.Oxidative stress by H2O2 induces DNA double strands break and the deletion of Hsp 90? causes DNA damage more serious.2.Oxidative stress by H2O2 can inhibit HepG2 Hsp90?+/+cells and HepG2 Hsp90?-/-cells,HepG2 Hsp90?-/-cells are more sensitive to H2O2 treatment.3.Oxidative stress by 500?M H2O2 caused G2/M assest and HepG2 Hsp90?-/-cells is more serious than HepG2 cells.4.Oxidative stress by H2O2 causes cell apoptosis and HepG2 Hsp90?-/-cells is higher than HepG2 cells of apoptotic rate.5.The level of MRN complex decreased under high concentration of H2O2,and the effect of low concentration H2O2 was not significant.6.Hsp90? interference,knockout,mutant plasmid transfer,inhibitors can lead to the reduction of MRN complex,and accompanied by H2O2 treatment of DNA damage increased and DNA repair reduced.7.RNA interference of MRN complex,the protein expression of y-H2AX was not obvious under oxidative stress by 500?M H2O2.8.Oxidative stress by H2O2 increased the foci formed of MRN complex and ?-H2AX at damage site,whereas the deletion of Hsp90a reduced the foci formation of the MRN complex and y-H2AX at damage site.Conclusions1.Oxidative stress by H2O2 induces DNA double strands break and the deletion of Hsp90? causes DNA damage more serious.2.Oxidative stress by H2O2 inhibits cells proliferation,causes cell cycle arrest and apoptosis,and the deletion of Hsp90? is more sensitive to the treatment of hydrogen peroxide,cell cycle arrest is more serious,the apoptosis rate increase.3.The level of MRN complex decreased under high concentration of H2O2,and the effect of low concentration H2O2 was not significant,suggesting that the background MRN complex level is more important for DNA damage repair.4.Hsp90? interference,knockout,mutant plasmid transfer,inhibitors can lead to the reduction of MRN complex,and accompanied by H2O2 treatment of DNA damage increased and DNA repair reduced.5.Under oxidative stress,Hsp90? knockout affects the function of MRN complex,which makes the ability to identify DNA double-strand breaks and DNA double-strand damage repair ability.