The Mechanism Of Regulating The Expression Of CCL17 By NOTCH1 Mutation In Chronic Lymphocytic Leukemia

BACKGROUND: Chronic lymphocytic leukemia(CLL)is a heterogeneous indolent B lymphocyte tumor which usually attacks the elderly.There are many signaling pathways involved in the occurrence and development of CLL.NOTCH1 is a ligand-dependent activated transcription factor,which plays an important role in the proliferation of lymphoid cells.However,the role of its mutants involved in the disease progress of CLL(e.g.Richter syndrome)remains unclear.OBJECTIVE: In this study,we constructed overexpression ICN(intracellur domain of NOTCH1)and CTdel(mutation ICN)plasmids,and established ICN unmutated and mutated cells to study its pathway activity and the related cytological functions in vitro,then to explore the mechanism of regulation of HDAC1/MTA2 by mutant ICN in molecular level.Therefore,we will reveal the mechanism of mutant ICN modulating HDAC1/MTA2 to stimulate the expression of CCL17 and accelerate Richter transformation of CLL,and explore the novel therapy target for CLL.METHODS:(1)The wild type plasmid expressing ICN and the CTdel ICN plasmid were constructed and electro-transferred into Ba F3 cells respectively.The m RNA and protein expression of ICN were verified by q RT-PCR and Western Blot assay.The experiment was divided into three groups,including the empty vector group,the overexpressing wild group and the mutant group.Then these plasmids were transfected into 293 T cells and the localization of ICN protein was detected by immunofluorescence assay.(2)CSL luciferase reporter plasmid was constructed to detect the transcriptional activity of NOTCH1 in each group.Transcriptome sequencing was performed to analyze the sequencing data by using bioinformatics analysis,including GO and KEGG methods.The expressions of CCL17 and CCL22 were also detected by q RT-PCR and ELISA.Transwell experiments were performed to detect the migration ability of thymic T cells induced with the supernatants in each group.(3)In order to further explore the mechanisms,mass spectrometry and Co-IP were performed respectively to detect the binding ability of ICN with MTA2/HDAC1 in each group.The protein domain of the NOTCH1 mutant portion which binded directly with MTA2/HDAC1 was confirmed by GST pull down assay.Finally,Ch IP was used to detect the binding abilities between MTA2/HDAC1 and the promoters of CCL17 in each group.RESULTS:(1)The expression of m RNA and their protein of ICN in the wild-type group and mutant group were significantly higher than that in the empty vector group.The size of ICN protein in the mutant group was smaller than that in the wild-type ICN group.ICN in the wild-type and mutant groups were localized in the nucleus according to the immunofluorescence.(2)The luciferase reporter system showed that both of the CSL luciferase activity of the wild-type and the mutant group were higher than that of the empty vector group,while the CSL luciferase activity of the mutant group was higher than that of the wild-type group.The results of q RT-PCR and WB showed that both of the expression of CCL17 in the wild-type group and the mutant group were higher than that in the empty vector group,while the expression of CCL17 in the mutant group was significantly higher than that in the wild-type group.The secretion of CCL17 protein in the mutant group cells was significantly increased according to ELISA results.Transwell test showed that the migration ability of thymic T cell increased significantly in the mutation group than that in the wild-type group.(3)Mass spectrometry and Co-IP showed that the wild-type ICN might bind with MTA2/HDAC1,but the ability of mutant ICN recruiting MTA2/HDAC1 decrease significantly.GST pull down assay showed that the protein domain of the C-terminal mutant portion of NOTCH1 binded directly with MTA2/HDAC1.Finally,the ability of MTA2/HDAC1 activated CCL17 promoters were significantly higher in the wide-type group than that in mutant group or in the empty vector group according to Ch IP assay.CONCLUSION: ICN binds to the negative transcription factor MTA2/HDAC1 in the nucleus and plays a balance function in the regulation of transcriptional activation of ICN/CSL/Co A which promotes the expression of the downstream genes.However,the ability of mutant ICN recruiting MTA2/HDAC1 decreases significantly due to the truncated C-terminus of ICN which locates in the nucleus.Thus,the negative regulation of MTA2/HDAC1 on the expression of CCL17 is inhibited,resulting in the up-regulation of CCL17,which causes migration of thymic T cells and promotes CLL cell proliferation.