The Role And Molecular Mechanism Of SPRDM16 Of SUMOylation In The Acute Myeloid Leukemia

Research Background:Acute myeloid leukemia, also named acute nonlymphocyticl eukemia(ANLL), is a kind of cancer from bone marrow blood cells. The main characteristic is that a rapid growth of abnormal white blood cells concentrates in bone marrow to disrupt the formation of normal blood cells. Acute myeloid leukemia is the most common acute leukemia among the adult, and its morbidity increases with age and develops rapidly. If the treatment not timely, it will cause a death within a few weeks. Already it has discovered many risk factors for leukemia disease due to the gene mutation and chromosome abnormality, but like other tumors, the specific pathogenesis of leukemia remains unknown. Except that genetically repeated abnormal chromatin and gene mutation is particularly important in the pathogenesis of leukemia, epigenetic regulation such as DNA methylation, histone modification and microRNA also plays an important role for AML. With a deepening study on the leukemia mechanism, now it is also found that protein post-translation in the process of the occurrence of leukemia present a diverse and important role. In recent years, a large number of articles further reveal that SUMOylation participates in the occurrence, development and outcome of leukemia positively and widely. Many studies have shown that PRDM16 functions as a very important transcription cofactors and an important node of its upstream and downstream genes, and also plays an important role for dry maintenance and differentiation of many cell types. Notable point is that PRDM16 has connected with hematopoietic system and leukemia, however, the exact function and significance of sPRDM16 SUMOylation on the acute myeloid leukemia development is still unknown.Research purposes:1.To explore biological phenotypic effects of sPRDM16 on acute myeloid leukemia;2.To investigate the role and regulation mechanism of sPRDM16 SUMOylation on promoting the activity of tumor;3.To verify clinical phenotype effects of sPRDM16 SUMOylation in mice and even patients with acute myeloid leukemia.4.To determine the regulation of sPRDM16 SUMOylation on the expression of its downstream differentiation related genes of acute myelogenous leukemia.Research methods:1.Exogenous transfection and co-immunoprecipitation assays were performed to verify sPRDM16 SUMOylation and its deSUMOylation, and sPRDM16- K568 R gene sequence was set up by one site mutation.2.Tumor relevant assays were carried out to validate that sPRDM16 was able to stimulate tumor activity: a proliferation assay was performed to study the effect of sPRDM16 SUMOylation on leukemia cell proliferation; in vitro soft agar clone formation assay was performed to study sPRDM16 SUMOylation for the anchor qualitative growth of leukemia cell clone formation ability; in vitro PMA induced differentiation assay was performed to explore the effects of sPRDM16 SUMOylation on acute leukemia differentiation abilities.3.Human acute myeloid leukemia transplantation mice model was established to explore the biological function of sPRDM16 SUMOylation in vivo, and even the relationship with clinical phenotypes.4.RNA – sequencing was used to search the effect of sPRDM16 SUMOylation on its downstream differentiation related genes and signaling pathways on large-scale.5.Quantitive Real- Time PCR test was used to confirm these target genes found by RNA-seq to verify this effect of s PRDM16 SUMOylation on its downstream signals.Research results:1.In vitro, both sPRDM16 and PRDM16 can be SUMOylated, SUMOylation is independent on its PR domain; K568 is its SUMOylation site; SENP1 can deSUMOylate sPRDM16.2.sPRDM16 can stimulate the activity of human acute myeloid leukemia tumor, increase its proliferation rate, enhance the ability of clone formation, and inhibit tendency of tumor differentiation.3. sPRDM16 SUMOylation is indispensable for tumor activity promotion, sPRDM16K568 R cannot be SUMOylated but reduce the clone forming ability, and rescue the differentiation ability of tumor cells to a certain extent.4. sPRDM16K568 R tumor cells seems to have faster proliferation in vitro. Furthermore, sPRDM16 SUMOylation impact on promoting tumor proliferation rate to some degree due to an integration of biological functions.5.sPRDM16 SUMOylation play a vital influence on human acute myeloid leukemia cells transplantation mice model phenotype. sPRDM16 SUMOylation pushes leukemia cells to grow faster, lost more weight, lower hemoglobin, and worse prognosis after transplantation. Differentiation capacity reduction may be the reason to cause these phenotypes.6. In the process of in vitro PMA induced differentiation, sPRDM16 WT and sPRDM16K568 R affect a series of related genes, such as KLF10, LIF, BCL3, HDAC9 and CCL5, IL6 R, NUMB, etc.Conclusion of this thesis:sPRDM16 has a vital function to enhance the activity of tumor cells in the occurrence of acute myeloid leukemia, and its SUMOylation is indispensable positively to impact on acute myeloid leukemia. A series of in vitro and in vivo studies verified that sPRDM16 SUMOylation affect the phenotype and prognosis of acute myeloid leukemia on multiple aspects. Therefore, we used RNA-seq to confirm that SUMOylated sPRDM16 influenced multiple differentiation related downstream gene such as KLF10, LIF, BCL3, HDAC9 and CCL5, IL6 R, NUMB, etc, it also showed that sPRDM16 SUMOylation may affect the multiple signaling pathways involved in cell differentiation.