The Role Of SUMO Modification Of HLTF In The Occurrence And Development Of Leukemia And The Study Of Its Molecular Mechanism

Leukemia is a severe clonal disease of the hematopoietic system characterized by dedifferentiation and malignant proliferation of immature hematopoietic precursor cells in the bone marrow.According to the Global Cancer Statistics 2018 published by the World Health Organization(WHO),the total number of leukemias in the world in 2018 is about 437 thousand killing about 309 thousand and the death rate is as high as 70.7%.The classification and prognosis of leukemia are very complex,and it is necessary to formulate specific treatment plans according to the specific leukemia types and prognosis of different patients.Therefore,there is no uniform and widely applicable treatment for leukemia treatment.The helicase-like transcription factor(HLTF)belongs to the SWI/SNF family and is a chromatin remodeling factor.It is involved in repair after DNA damage,and its down-regulation increases gene mutations and chromosomal abnormalities.Current research indicates that HLTF is associated with the production and development of acute myeloid leukemia(AML),but its specific mechanism of action remains unclear.SUMOylation is an ubiquitin-like modification catalyzed by SUMO-specific activation enzyme(El),binding enzyme(E2),and ligase(E3).It is covalently linked to substrate proteins,regulating the localization of target proteins and the interaction of target proteins with other biomacromolecules.According to software predictions,HLTF proteins are likely to undergo SUMOylation.This thesis mainly studies the effects of HLTF and its SUMOylation on leukemia cells and the molecular mechanism of SUMOylcation of HLTF in the pathogenesis and progression of leukemia cells.Based on the experimental techniques of vector construction,lentiviral packaging and infection,we knocked down or overexpressed HLTF in leukemia cells,and detected the proliferative capacity of leukemia cells by cell counting.The results showed that the proliferation ability of leukemia cells in HLTF knockdown group was significantly lower than that.In the control group,the proliferative capacity of leukemia cells in the HLTF overexpressing group was significantly higher than that in the control group.Using the differentiation inducer PMA induction and real-time quantitative PCR to detect the mRNA expression level of leukemia cell differentiation marker CD11b,it was found that the differentiation ability of leukemia cells in HLTF knockdown group was significantly higher than that in the control group,while the differentiation ability of leukemia cells in HLTF overexpression group was significant lower than the control group.These results indicate that HLTF can promote the proliferation of leukemia cells,inhibit the differentiation of leukemia cells,and play an oncogene role in leukemia cells.We predict that HLTF may be modified by SUMO.HLTF was mainly modified by SUM02 by protein immunoprecipitation.Immunofluorescence experiments showed that HLTF and SUM02 and E2 binding enzyme UBC9 colocalized in the nucleus,further verifying the interaction between HLTF and SUM02.We detected that the E3 ligase that mediates HLTF SUMOylation is mainly PIAS2a by protein immunoprecipitation.Immunofluorescence experiments show that HLTF and PIAS2a colocalize in the nucleus,while the specific protease that mediates HLTF de-SUMOylation is mainly SENP2.We further utilized the point mutation vector construction and protein immunoprecipitation experiments to confirm that the SUMOylation site of HLTF is the 515th lysine residue(K515).These results show that HLTF completes SUMOylation modification by PIAS2a and de-SUMO modification by SENP2,and its SUMOylation site is K515.It was detected by protein immunoprecipitation that SUMOylation can promote the ubiquitination degradation of HLTF and thus reduce the stability of HLTF.We also examined the role of PML in SUMOylation of HLTF,and found that PML can promote SUMO of HLTF.Resulting in HLTF degradation by ubiquitination pathway,immunofluorescence experiments show that HLTF and PML colocalize in the nucleus.The results of protein immunoprecipitation experiments showed that RNF4 can promote ubiquitination degradation of HLTF as HLTF E3 ubiquitin ligase.Treatment with arsenic trioxide(As2O3)induced the expression of SUM02 in leukemia cells,and the SUMOylation of HLTF increased,resulting in decreased HLTF stability.SUMOylation can reduce the stability of HLTF by promoting ubiquitination degradation of HLTF,and HLTF acts as an oncogene in leukemia cells,so SUMOylation of HLTF inhibits the development of leukemia by reducing its stability.In summary,this study investigated the role and molecular mechanism of SUMOylation of HLTF in leukemia,and found that SUMOylation can promote the ubiquitination of HLTF,reduce the expression of HLTF,and inhibit the proliferation of leukemia cells,but promote differentiation of leukemia cells,RNF4 can act as an E3 ubiquitin ligase of HLTF to promote ubiquitination degradation of HLTF.Our research further found that both PML and arsenic trioxide can promote SUMOylation of HLTF and promote the differentiation of leukemia cells.The results of this paper lay a good foundation for further understanding of the role and molecular mechanism of SUMOylation of HLTF in the pathogenesis and progression of leukemia,in order to find key signaling pathways and molecules,thus providing new treatment target for clinical treatment of leukemia.