Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.

Objective:To identify the differentially expressed snoRNAs in the carcinogenesis of cells induced by α-particles radiation and predict the targeted genes and RNA-co-expression networks.Methods:Full transcriptome expression microarray biochips were employed to screen the differentially expressed snoRNAs between human bronchial epithelial BEP2D cell line and its derivative malignantly transformed cell line BERP35T-4 established by α-particle irradiation. The expression changes of snoRNAs and their derived sdRNAs were confirmed by qRT-PCR. The functional domains, targets and co-expression networks of snoRNA were predicted by bioinformatics analysis.Results:Consistent with the result of microarray assay, the expression changes of the screened snoRNAs were confirmed by qRT-PCR. The expressions of sno116 family decreased in BERP35T-4, which was 0.105% ( t=26.60, P<0.01) of BEP2D, and they were generally down-regulated in radiation-induced carcinogenic BERP35T-4 cells and the human lung cancer cell lines A549 and H1299. It was also found that the expression level of the sdRNAs derived from sno116-14 was significantly different in the same cells. It was speculated that these less expressed sdRNAs of sno116-14 could be due to degradation as the consequence of interaction with their targets. The co-expression networks of sno116 family with other types of RNA were established, and the predicted targets of sno116-14 included ZNF280D, TFDP1, CCDC28B, RPS6KA3, CANX, RUNX1 and KALRN, which were related to the functions of cell proliferation and cytoskeletal structure. Conclusions:Some differentially expressed snoRNAs related to α-particle induced carcinogenesis have been identified. It is predicted that the target gene of sno116-14 is involved in the biological processes such as cell proliferation, cytoskeletal structure and the signaling pathways for function regulation, providing new information for the function model of C/D box snoRNAs and the mechanism of radiation carcinogenesis.

Objective: To identify the differentially expressed miRNAs in the exosomes secreted from γ-ray irradiated cells and provide new clues in disclosing the mechanisms of radiation-induced bystander effects. Methods: The human bronchial epithelial cells (BEP2D) were irradiated with ⁶⁰Co γ-rays, and the exosomes were collected by ultracentrifugation from the culture medium of 2 Gy-irradiated cells and non-irradiated control. The exosomes were identified by an electron microscopy. The miRNA microarray technique was used to analyze the miRNA expression profiles in the exosomes. qRT-PCR was used to verify the miRNAs expression. The functional pathways of miRNAs targeting genes were predicted by informatic analysis using the databases of TargetScan, miRanda, GO and KEGG. Results: Sixteen miRNA with significantly increased expression (P<0.05) were identified in the exosomes of BEP2D cells at 4 h post-2 Gy irradiation as compared with the non-irradiated control cells, among which miR-100-5p, miR-1246, miR-29b-3p, and miR-7-5p were further confirmed to be unregulated by qRT-PCR assay (P<0.05). Meanwhile, the expression changes of above-mentioned four miRNAs were also investigated in the irradiated cells. The data indicated the expression was significantly increased at 2 h post-2 Gy irradiation for the miR-100-5p, miR-1246, miR-29b-3p in addition to miR-7-5p. However, all these four miRNAs were downregulated in the cells at 4 h post-irradiation and then gradually recovered. Bioinformatics analysis showed that the targeted genes of these differentially expressed miRNAs might participate in the biological processes and signal pathways of cell adhesion, mTOR signal pathway, chromatin modification, HR and NHEJ pathways of DNA repair and so on. Conclusions Radiation-inducible miRNAs have been identified in the exosomes from the irradiated BEP2D cells. The target genes of these miRNAs play roles in a series of important biological processes and functional pathways, which provides new clues in elucidating the mechanisms of radiation-induced bystander effects.

Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.

Objective To study the changes in miRNAs expression in the exosomes of human umbilical vein endothelial cells(HUVECs) after 60 Co γ-rays expose using microRNA(miRNA) chips and bioinformatics techniques so as to provide new clues to the mechanism of radiation-induced vascular tissue injury and its bystander effects.Methods HUVECs exosomes were collected in the control and 4 Gy irradiated cells by ultra-high-speed centrifugation,and further confirmed using transmission electron microscopy (TEM) and Western blotting of exosomes biomarkers.miRNA microarray was used to analyze miRNA expression profiles of exosomes and cells.Also,real-time quantitative PCR(qRT-PCR) was used to verify differentially expressed miRNAs,and the miRDB and TargetScan were performed to predict the target genes of the differentially expressed miRNAs.Bioinformatics analysis was performed using DAVID,KEGG and other online tools.Results Compared with the control exosomes from non-irradiated HUVECs,miRNA microarray analysis revealed that 5 up-regulated,and 13 down-regulated miRNAs were identified in the exosomes from HUVECs at 0.5 h after 4 Gy-irradiation,and 16 up-regulated and 5 down-regulated miRNAs at 2 h after 4 Gy-irradiation.Moreover,38 and 85 miRNAs were differentially expressed respectively in the HUVECs at 0.5 h and 2 h after radiation.The difference was statistically significant(P<0.01).The results of bioinformatics showed that these miRNAs might exert the radiation-induced bystander effect (RIBE) by regulating MAPK signal pathways,RAS and PI3K-Akt signal pathways.Conclusion The ionizing radiation injury significantly alters the components and expression levels of exosomal miRNAs,which play important roles in regulating the signal pathways in response to radiation.

Objective To investigate the expression of radiation-induced IER5 protein and screen its potential interaction proteins that may participate in DNA repair process.Methods HeLa cells were irradiated with 4 Gy ionizing radiation.IER5 protein expression in whole cell lysate and in nuclear fraction were detected by Western blot at different timepoints after irradiation.3 × Flag-IER5 pCMV plasmid was constrcuted and the Flag tagged-IER5 expression was verified by Western blot.293T cells were transfected with 3 × Flag-IER5 pCMV plasmid.After irradiation the cells were collected and proteins were extracted.The IER5 interaction proteins were purified using immunoprecipitation and separated by 12％ SDS-polyacrylamide gel electrophoresis.Then the binding proteins were cut from the gel and analyzed by Mass spectrometry.Results The expression of IER5 protein began to increase 4 hour post-irradiation and its peak level was observed at 12 hour post-irradiation,and it lasted until 48 hour after irradiation.The expression level of IER5 protein in whole cell lysate and nuclear fraction were both increased.With the mass spectrometry analysis,a total of 347 proteins and 256 proteins were identified in irradiated and nonirradiated groups,respectively.Fourty one differential proteins were obtained,where 10 proteins were associated with DNA metabolic process and DNA rapair in the irradiated group and the poly(ADP-ribose) polymerase 1 (PARP1) protein was further confirmed by Western blot.Conclusions IER5 protein is an DNA damage related protein,and it may participate in DNA repair process.

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.

Objective To determine the Golgi dispersal in radiation damaged cells and the protective effect of vanillin derivatives.Methods Immunofluorescence, cell cycle analysis of flow-cytometry,Western blot,and clone formation were used.Results Immunofluorescence observation showed that the Golgi dispersal caused by 2 Gy 60 Coγ-ray was significantly increased in a dose-dependent manner in the range of 4-10 Gy as was demonstrated by the fact that the Golgi area was significantly increased. When the irradiated cells were treated with the radioprotective agent VND3207, a vanillin derivative,the Golgi dispersal induced by radiation was significantly reduced.The radiation-induced Golgi dispersal was also displayed in a pattern of time-course after irradiation in the HeLa cells, and persisted at least to 36 h post-irradiation. Cell cycle test results indicated that the Golgi dispersal was not associated with the G2/M arrest triggered by radiation-induced DNA damage response.VND3207 could promote cell survival by plate colony formation assay.Conclusion The Golgi dispersal can be caused byγ-ray irradiation in a dose-and time-dependent manner, and VND3207 can provide a good protection against radiation injury associated with inhibited Golgi dispersal.

In the early 1950s,China′s modern industry and national defense developed rapidly. While toxicological research,represented by occupational toxicology,radiological toxicology and military toxicology,was initiated. Along with the expansion of industrial and agricultural activities and production, high- speed economic development,and the changes of the environment and lifestyles,the objects and content of toxicological research have also been expanded. Subdisciplines of toxicology have kept emerging,and an integrated toxicological discipline and research system has been established. Toxi?cological research has become an active area in China. Toxicological publications from China have accounted for about 10% to 20% of total international toxicological research papers in the recent ten years. Toxicologists in China have paid much attention to the toxicities and biological safety of new materials and emerging pollutants,such as nanomaterials and fine particles in the air. Some toxicological research was discussed in this paper.

Objective To evaluate the radiosensitization and mechanism of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 on HCT116 colorectal cancer stem cells.Methods The flow cytometry was used to determine the sub-population of cancer stem cells with the markers CD133/ CD44.The cells were divided into four groups:control group,20 μmol/L NU7926 group,2 Gy irradiation group,and 20 μmol/L NU7026 combined with 2 Gy irradiation group.Cell proliferation and survival were evaluated by colony-formation experiment.Flow cytometry was used to analyze the cell cycle distribution and apoptosis induction.γ-H2AX foci were detected with immune-fluorescence staining analysis by a laser confocal microscopy for investiating the DNA double-strand break repair.Results The flow cytometric data of CD133 +/CD44 + positive cells indicated that the sub-population of cancer stem cell (CSC) took the ratio of (88.14 ± 0.47)％ of the cultured HCT116 cells.The colony-formation efficiency of HCT116 cells was (84.75 ± 1.35) ％ in serum-free mediums in vitro culture.Compared to 2 Gy irradiation alone group,the NU7026 combined with 2 Gy irradiation group had a lower cell colony-forming ratio (t =7.21,P ＜0.01) and a lower survival ratio (t =7.22,P ＜ 0.01).The proportion of CSCs sub-population increased at 48 h post-2 Gy irradiation,suggesting that HCT116 CSCs was more resistant to ionizing radiation.Importantly,NU7026 largely decreased the proportion of CSCs sub-population in 2 Gy-irradiated cells.The difference of CSC proportion between 2 Gy irradiation alone group and 2 Gy combined with NU7026 treatment group was statistically significant (t =9.55,P ＜ 0.01).In addition,the group of NU7026 combined with 2 Gy irradiation had a higher ratio of G2/M arrest 24 h post-irradiation (t =7.67,P ＜ 0.01) and an increased induction of cell early apoptosis (t =8.24,P ＜ 0.05).48 h post irradiation as compared to 2 Gy irradiation alone group.NU7026 treatment significantly inhibited the cellular capacity of repairing DNA double-strand breaks induced by γ-ray irradiation.The γ-H2AX foci of the combined treatments group were much higher than that of 2 Gy irradiation alone group at 2,4,8,24 h postirradiation (t=19.58,11.95,7.01,9.45,P＜0.01).Conclusions DNA-dependent protein kinase catalytic subunit inhibitor NU7026 can significantly sensitize the cancer stem cells of colorectal carcinoma HCT116 cells to γ-ray irradiation.Multiple mechanisms are involved in the radiosensitization effect of NU7026,including DNA repair inhibition,elongation of G2/M arrest,and increase of radiation-induced apoptosis.