Alteration Of AP1S1 Expression In AHH-1 And Its Bystander Cells Irradiated By ⁶⁰Coγ-ray And Its Role Research

Radiation kill nomal and tumor cells mainly through inducing target cells DNA and biological macromolecules damage.At the time of DNA being damaged directly, intercellular signal transduction, that is, gap junctions and inflammation-mediated bystander effect in the cell, play an important role in the organization's response to chemotherapy and radiation. Radiation-induced bystander effect means indirectly-irradiated cells have the analogical biological reaction with directly-irradiated cells.For example, chromosome aberration, genomic instability, uncontrolled cell growth, signal transduction change et al.The bystander effect becomes a hotpoint study on low dose effect, however, until now the reason,mechanism and endpoint of the bystander effect are still unsure.The bystander effect involved a large number of gene expression changes, therefore, gene expression change is an important feature of the radiation induced bystander effect. It is significant that we may explore the mechanism of radiation bystander effect by studying some of the differential expression genes in the bystander effects.The AP1S1 (adaptor-related protein complex 1, sigma 1 subunit) is part of the clathrin coat assembly complex which links clathrin to receptors in coated vesicles. These vesicles are involved in endocytosis and Golgi processing. The gene is a differential expression gene in our microarray which screens differential expresstion genes of radiation induced bysander effect. The microarray results show that AP1S1 gene expression highly increased in the bystander cells (P<0.05). Because the bystander effects partly induced by the transmission of signaling molecules among the cells, moreover, the gene in the cell also play an important role in endocytosis. Therefore, based on the differential expression of AP1S1 in AHH-1 and its bystander cell post-radiation, we want to study the role of AP1S1 gene in radiation induced cell damage and the bystander effects indirectly.Constent of study1. Sdudy the bystander effect of AHH-1 induced by different dose of 60Coγ-ray in the induction rate of MN, cell proliferation, cell cycle,and the mRNA and protein expression of AP1S1 gene at different time post-radiation.2. Interfere the AP1S1 gene expression of AHH-1 cell with AP1S1 siRNA, illuminates the role of AP1S1 expression on radiation-induced bystander effect, makes out whether it can protect the injury induced by bystander effect that the AP1S1 gene expression of AHH-1 cell being inhibited.Expremental methods1. Preparation of bystander cell model2. Investigate the bystander effect of AHH-1 cell with the induction rate of MN, the cell proliferation and cell cycle. RT-PCR and Western Blot were performed to determine the mRNA and protein levels of AP1S1 gene respectively at 8,24h post-radiation.3. Design the AP1S1 siRNA, transfect the AHH-1 cell, Verify the transfection efficiency with RT-PCR and Western Blot.4. The transfected AHH-1 cells which co-culture with namal AHH-1 cell exposed with different dose of 60Coγ-ray is considered as bystander cells group, negative control group and nontransfected group are the control group. Investigate the induction rate of MN, the cell proliferation, cell cycle.5. Statistics method:process data with SPSF13.0 statistical software.Results1. Result of verifying microarray:the mRNA and protein level of AP1S1 in the directly irradiated cells and bystander cells upregulated in different time point compared with the control groups with a dose dependant manner. In addiation, the bystander cells upregulated significantly.2. Result of siRNA interference:the siRNA AP1S1-1's transfection efficiency is high, AP1S1-2 followed the AP1S1, AP1S1-3's transfection efficiency is lowest.3. Result of cell proliferation used CCK-8:AP1S1-1 transfected group has a higher transfection efficiency, the corresponding rate of cell proliferation were higher than the negative control and non-transfected group (P<0.05); AP1S1-2 transfected group followed the transfection efficiency, cell proliferation rate is higher than negative control group and non-transfected group, but P> 0.05, no statistical significance;AP1S1-3 transfected group, its transfection efficiency is low, no statistical significance.4. Bystander cells culture medium levels of NO determination results. NO levels of each group changed little, P> 0.05, no statistical significance.5. Result of MN:AP1S1-1 transfected group has a higher transfection efficiency, the corresponding rate of MN were lower than the negative control and non-transfected group (P<0.05); AP1S1-2, AP1S1-3 transfected group, its transfection efficiency is poor, no statistical significance. 6. Cell cycle result:nomal bystander cells show G2 arrest; AP1S1-1 group has a higher G2 arrest compared with negative control group.Conclusion1. AP1S1 gene expression upregulated in the bystander cells, maybe it plays a important role in radiation-induced bystander effect.2. Induction rate of MN reduced and cell proliferation increased after AP1S1 gene expression interfered, it shows AP1S1 has a contribution to bystander effect damage at least.3. Cell culture medium NO levels does not change significantly, indicating AP1S1 gene in bystander effect of ionizing radiation signal membrane transport may play a role, but inhibition expression of AP1S1 can not reduce the bystander effects cell NO generation.4. Bystander effect in nomal AHH-1 cell has a G2 arrest phenomenon, it is similar with directly irradated cell; the transfected cell has a higher G2 arrest than the corresponding negative group, it show AP1S1 gene expression down-regulation can improve the G2 arrest, thereby promote the DNA damage repair before the G2/M checkpoint...