The Mechanism Of 53BP1 Regulated By SET-mediated Histone Modification In Cr(?)-associated Carcinogenesis Based On Quantitative Proteomics

Hexavalent chromium(Cr(?))is a common commerical material that has been widely used in manufacturing and processing industries,such as electroplating and dyeing,which can significantly increase risk of lung cancer followed by long-term exposure.However,the specific molecular mechanism of Cr(?)-induced lung cancer is still unclear.Our previous studies have found that long-term low-dose Cr(?)exposure induced DNA damage and malignant transformation in 16HBE(16HBE-T)cells,while 53BP1 expression,a key mediator of DNA repair was down-regulated and histone modification was involved in that process.However,the role of association between 53BP1 and histone modifications and upsteam molecules in the carcinogenesis of Cr(?)has not been studied.In this study,we used quantitative proteomics method to construct the differential protein expression profile and histone modification profile of 16HBE-T cells,and verified the role of histone modification mediated by SET prorein in the regulation of 53BP1.The study aims to reveals the potential molecular mechanism of Cr(?)carcinogenesis from the aspects of gene and epigenetics,providing theoretical and experimental basis for early warning prediction and discovery of tumor therapeutic targets.The research works were carries out from the following aspects.Part 1:Construction of differential protein expression profile and histone modification profile of Cr(?)-induced malignant transformation 16HBE cells(16HBE-T)based on quantitative proteomics.Methods:Total protein and histone were collected from malignantly transformed 16HBE cells(16HBE-T)and 16HBE cells.Quantitative proteomics method was used to screen and identify differential proteins and histone modification,which were confirmed by Western blot.Results:A total of 3517 proteins were found,the expression differences greater than 1.5 or less than 0.67 times were to found have 185 and 201 proteins,respectively.Cluster analysis revealed that differential proteins were mainly involved in DNA damage repair,autophagy,RNA processing and other biological processes.Western blot results showed the expression level of SET,PKACA were significantly increased and CaMKK2 was decreased significantly.Meanwhile,histone H3K18 and H3K27 acetylation,two key modification marks were identified and decreased significantly campared to control group(P<0.05).Conclusion:Alteration of proteins involved in multiple biological processes and molecular functions were found in 16HBE-T cells.At the same time,the levels of histone H3K18ac and H3K27ac were significantly decreased.Part 2:The role of 53BP1 regulated by histone H3K18ac and H3K27ac mediated by SET in Cr(?)-associated carcinogenesis.Methods:16HBE-T and 16HBE cells were collected.The H3K18 and H3K27 acetylation within the promoter region of 53BP1 were measured by chromatin immunoprecipitation-quantitative PCR(ChIP-qPCR).The level of 53BP1,histone H3K18ac and H3K27ac were analyzed after inhibiting SET through siRNA.The variation of cell cycle and apoptosis was evaluated by using flow cytometry.Moreover,Western blot was used to detect the expression level of SET,H3K18ac,H3K27ac and 53BP1 in different lung cancer cells.Results:The modification level of H3K18ac and H3K27ac in promoter of 53BP1 were significantly decreased in 16HBE-T cells by comparing control group cells.Not only the global level of H3K18ac,H3K27ac and 53BP1 but also the level of apoptosis were differentially increased in 16HBE-T cell after inhibiting SET.At the same time,cell cycle were arrested at phase of G1/G0.Besides,the results of Western blot suggested that the regulatory mode of acetylation-regulated interaction between 53BP1 and H3K18ac,H3K27ac mediated by SET was unconsistent with other lung cancer cells.Conclusion:The modification level of H3K18ac and H3K27ac mediated by SET could furtherly affected the transcription of 53BP1.The level of apoptosis was significantly increased and cell proliferative potential was inhibited after knockdown of SET.However,the regulatory mode of 53BP1 through H3K18ac and H3K27ac mediated by SET was unconsistent with other lung cancer cells.Part 3:The expression levels of SET,H3K18ac,H3K27ac and 53BP1 in SD rat exposed to Cr(?)in vivoMethods:The in vivo exposure assay of SD rats was established by intrathoracic injection.After one year,the rat was sacrificed and lung tissues were dissected and obtained.The expression levels of SET,H3K18ac,H3K27ac and 53BP1 were detected by Western blot.Results:Histopathological results showed that inflammatory cells infiltrated in the lung tissue but none visible tumor lesion in resetecd lung tissue was found by naked eye,and no obvious cancer nest was observed.Western blot analysis showed that the levels of H3K18ac and H3K27ac were significantly decreased,while the expression of SET and 53BP1 did not significantly change.Conclusion:The levels of H3K18ac and H3K27ac were significantly decreased,which may be changed prior to genntic alterations and exist at the early stage of tumorigenesis.