Regulation Mechanism Of Histone Related To Inhibition Of 53BP1 In Cr(?)-induced DNA Damage

Chromium pollution is one of the world's major environmental problems which can not be solved at present,and the chromate industry has long been ranked in the top of the serious polluting industries in our country.Epidemiological investigation shows that long-term chromium exposure can significantly increase the incidence of lung cancer.53BP1 as a tumor suppressor gene p53 binding protein,is one of the key markers of DNA damage repair,its abnormal expression is related to the occurrence and development of tumor.We found that the acute exposure of hexavalent chromium[Cr(?)]could cause significant DNA damage and inhibited the expression of 53BP1.In this study,we combine the toxicology research method and proteomics technology to investigate the interaction between histone modification and 53BP1 in Cr(?)-induced DNA damage and preliminarily elucidate the toxic and molecular mechanisms of Cr(?),which provided a new idea for the development of preventive diagnosis and treatment strategy of occupational lung cancer.The project includes three parts as follows:1.Screening and verification of the effect of histone acetylation modification on 53BP1 after Cr(VI)acute exposure16HBE cells were incubated with 0 ?mol/L and 5 ?mol/L Cr(?)for 24 hours.Histone was extracted by acid method,separated by biphasic gel,and identified and analyzed by mass spectrometry.The expression of key histone was verified by Western blot,and the regulatory mechanism of 53 BP 1 and key histone was analyzed by chromatin immunoprecipitation(CHIP).The results of mass spectrometry showed that there were different histone modifications on H2A,H3 and H4.Furthermore,two key proteins was found:H3K18ac and H3K27ac.CHIP showed that the levels of H3K18ac and H3K27ac in the promoter region of 53BP1 were decreased,suggesting that the inhibition of the expression of 53BP1 in DNA damage induced by Cr(?)may be related to the down-regulation of H3K18ac and H3K27ac.2.Studies on the DNA damage in the bronchial epithelial cell model of Malignant Transformation induced by Cr(?)16HBE were treated with different concentration of Cr(?)(0,0.625,1.25,2.5?mol/L)for 15 weeks.The malignant degrees of transformed cells were identified by the assays for anchorage-independent growth and tumorigenicity.According to the single cell gel electrophoresis(SCGE)assay,the DNA damage rate was calculated.The expression level of 53 BPl was determined by Western blot.The results of soft agar cloning and tumorigenesis in nude mice showed the 16HBE was malignant transformation after exposure to 2.5 ?mol/L Cr(?)for 15 weeks.Further migration and invasion experiments showed that the transformed cells had the characteristics of tumor cells.The results of single cell gel electrophoresis showed Cr(?)can cause DNA damage in different degree under chronic exposure.The results of Western blot showed that the relative expression of 53BP1 group was significantly lower than the control group in the 2.5 ?mol/L,and the difference was statistically significant(p<0.01).The results showed that chronic exposure to Cr(?)could cause obvious DNA damage and could induce cell malignant transformation by inhibiting the expression of 53BP1 which resulting in the failure to repair the DNA damage in time.3.Changes of histone acetylation in the process of cell malignant transformation16HBE were treated with different concentration of Cr(?)for 15 weeks.The levels of H3K18ac and H3K27ac were detected by Western blot and cell immunofluorescence.The results of Western blot showed that the level of H3K18ac and H3K27ac was significantly decreased in the cells treated with chronic exposure for 15 weeks,which was consistent with the results of acute exposure.The results of cellular immunofluorescence showed that H3K18 and H3K27 were mainly distributed in the nucleus,the protein content in the nucleus decreased and the fluorescence brightness decreased significantly,which was consistent with the results of Western blot.It is speculated that Cr(?)could inhibit the expression of 53BP1 by affecting the level of histone acetylation,and the damage of 53BP1 could not be repaired in time.