| Objective1.To explore the role of deubiquitinase BRCC3 in aGVHD after allo-HSCT.2.To investigate the molecular mechanism of deubiquitinase BRCC3 in aGVHD after allo-HSCT.3.To evaluate the role of deubiquitinase BRCC3 in GVL after allo-HSCT.Methods1.Isolate the kidney,liver,lung,and spleen from mice receiving syngeneic and allogeneic HSCT and evaluate the relative expression of BRCC3 after transplantation.2.Construction of the minicircle-BRCC3 plasmid which can stably express in vivo.3.Establish the aGVHD mice and compare the minicircle-BRCC3 group and control group:1)The survival,body weight changes and aGVHD scores were monitored and recorded.2)The target organs of mice,including skin,liver,lung and intestine,were collected for histopathological examination by H&E staining.3)The proportions and number count of different lymphocytes were measured by flow cytometry.4)The levels of cytokines secreted by T cells,including IFNγ,TNFα,IL-17A,IL-4,IL-10 and so on,were measured by flow cytometry.5)The target organs of mice were collected to further measure the relative expression of IFNβ by Realtime PCR.4.Identify the potential substrate of BRCC3 by Ubiquitinomics Sequencing and Immunoprecipitation-Mass Spectrometry(IP-MS).5.Western Blot was used to further estimate the expression of substrate protein candidates,including 14-3-3,TPP2 and Trim25 after knocking down BRCC3.6.Co-Immunoprecipitation and Western Blot were used to analyze the interaction between BRCC3 and the E3 ligase Trim25 in the transfected cells.The function of deubiquitinase BRCC3 in regulating of ubiquitination and expression of Trim25 were also estimated.7.Dual-luciferase Reporter Assay System and the Realtime PCR were used to measure the function of deubiquitinase BRCC3 in the transcription of IFNβ as well as the levels of cytokines,including IL-6,IL-1β and IL-12P40.8.Establish the aGVHD/GVL mice by transferring A20-luciferase,and compare the minicircle-BRCC3 group and control group:1)The survival,body weight changes and aGVHD scores were monitored and recorded.2)The growth of A20-luciferase cells in mice was detected by IVIS 100 Bioluminescent Imaging SystemResults1.The syngeneic and allogeneic HSCT mice were successfully constructed.The expression of BRCC3 was down-regulated after allo-HSCT.2.The minicircle-BRCC3 plasmid was successfully constructed and expressed.3.The aGVHD mice were successfully constructed.BRCC3 significantly attenuated aGVHD by comparing the minicircle-BRCC3 group with control group:a)The longer survival,lower weight-loss and aGVHD scores were observed.b)Reduced histopathological damage in skin,lung and small intestine were observed.c)The proportion of macrophages,the proportions and number count of donor-derived activated T cells(CD69+CD4+/CD69+CD8+)were significantly decreased.d)The production of IFNγ in CD4+/CD8+T cells were significantly inhibited.e)The production of IFNβ was up-regulated in target organs.4.58 proteins were deubiquitinated and associated with BRCC3 by Ubiquitinomics Sequencing and IP-MS.5.Knocking down BRCC3 decreased the protein levels of Trim25 while not affected the protein levels of 14-3-3 and TPP2 in the transfected 293T cells.6.BRCC3 interacts with and deubiquitinates the E3 ligase Trim25.7.Knocking down BRCC3 down-regulated the transcription of IFNβ in the transfected cells.On the contrary,over-expressing BRCC3 up-regulated the transcription of IFNβ while inhibiting the expression of proinflammatory factors,including IL-6,IL-1β and IL-12P40 in the transfected Raw264.7 cells.8.The aGVHD/GVL mice were successfully constructed.BRCC3 significantly attenuated aGVHD while retained GVL by comparing the minicircle-BRCC3 group with control group:a)The longer survival,lower weight-loss and aGVHD scores were observed.b)The growth of A20-luciferase was inhibited in vivo.Conclusion1.Over-expression of BRCC3 attenuates the severity of aGVHD.2.BRCC3 reduces macrophages frequency,inhibits the activation of donor T cells and the secretion of IFNy in T cells.3.BRCC3 interacts with,deubiquitinates and up-regulates the E3 ligase Trim25,thus promoting the production of IFNβ.4.Over-expression of BRCC3 attenuates the severity of aGVHD while retaining GVL effect. |
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