Research On The Role And Mechanism Of SIRT1 In Myelodysplasia Syndrome

Research backgroundMyelodysplastic syndrome(MDS),a group of clonal hematopoietic stem cell disorders,is characterized by bone marrow dysplasia,peripheral blood cytopenias and a high risk of transformation to acute myeloid leukemia.MDS usually suffered old people,with a median age at diagnosis of 71-76 years.Its incidence is between 5.3 and 13.1 cases per 100,000 persons.Due to an aging population,improving survival following treatments for other tumors that leave patients at high risk for subsequent development of therapy-related MDS(t-MDS),the incidence of MDS is increasing over time.The pathogenesis of MDS is complicated,currently is believed to be associated with epigenetic regulation,spliceosome-associated genes mutation and chromosomal genetic variation.Due to the advanced age of MDS patients,the proportion of allogeneic hematopoietic stem cell transplantation was very low.Non-transplant therapy still remains the dominant option.However,conventional chemotherapy has a large toxicity in elderly patients with MDS,and the patient has low tolerance.Therefore,the research on MDS targeted therapy is still to be improved.The nicotinamide adenine dinucleotide(NAD)dependent deacetylase SIRT1 is a well-studied sirtuin that deacetylates histones and a variety of non-histone proteins,thereby regulates activities including cell growth,survival,metabolism,senescence and carcinogenesis.There has been great controversy about the function of SIRTI in tumor development.SIRT1 function in cancer is apparently context-dependent.Different functions exist in different types of tumors,to promote or prevent carcinogenesis.For example,SIRTI high expression in fibrosarcomas inhibits tumor proliferation by deacetylation of HIF1a.While in other tumors,such as chronic myeloid leukemia and BRCA1 related breast cancer,SIRT1 high expression promotes cancer.Thus,the function of SIRT1 differs among different tumors.However,the function of SIRT1 in MDS disease has not been reported until now.Research methodIn this study,the expression of SIRT1 in primary MDS patients was detected by flow and western blotting.The effect on MDS cells was verified by cell proliferation assay,clone formation assay and mouse transplantation assay.By performing immune precipitation assay combined with mass spectrometry,enzyme-linked immunosorbent assay,siRNA library screening,proximity ligation assay(PLA),domain mapping assay,Electrophoretic Mobility Shift assay(EMSA),we explore the downstream mechanism of SIRT1.Finally,anti-MDS proliferation function of SIRT1 agonist SRT1720 was verified on MDSL,primary MDS cell and NHD13(NHD13)MDS transgenic mice model.Research result1.Low expression of SIRT1 in MDS promoting MDS proliferationFirstly,the expression of SIRT1 was detected in the original MDS patients by flow and western blotting.The results showed that SRT1 was low expressed in the primary MDS patients compared to normal controls.Secondly,to further explore the function of SIRT1 in MDS cells,we overexpressed SIRT1 in human MDS cell line MDSL,which promotes cell differentiation,increases cell apoptosis and inhibits clone formation and cell proliferation.Knockdown of SIRT1 expression in MDSL cell lines promotes its clone formation ability.The clone formation ability is weakened after SIRT1 overexpression in primary MDS cells rather than normal control cells.In vivo mouse assays showed that SIRT1 overexpression inhibits the transplanting ability of MDSL cell lines and prolongs the overall survival time.Correspondingly,SIRT1 downregulation promotes in vivo engraftment ability.2.Low expression of SIRT1 promotes MDS cells proliferation by increasing TET2 acetylation.To explore SIRT1 downstream target,we first detected SIRT1 catalyzed acetylation proteins in MDSL cells by immune precipitation combined with mass spectrometry,then performed SiRNA library screening in the top 32 genes through SIRT1 agonist SRT1720 treatment.The results showed that TET2 knockdown could significantly inhibit the tumor suppressive function of SRT1720 and SIRT1 overexpression in MDSL.In order to further study whether TET2 plays the downstream function by interacting with SIRT1,we conducted the immune precipitation assay and the PLA assay in the primary MDS cells.The results showed the interaction between SIRT1 and TET2.The domain mapping assay confirmed that SIRT1 mainly interacts with the TET2 c-terminal catalytic domain.Because SIRT1 is a deacetylase,we speculate that SIRT1 may function by 'interacting with and catalyzing TET2.Therefore,we further carried out the immune precipitation experiment,which proved that SRT1720 and SIRT1 overexpression decreases TET2 acetylation level.Correspondingly,due to low SIRT1 expression in the primary MDS cells,TET2 acetylation level was significantly higher than that in the normal control.Then we further detected the specific acetylation sites of TET2 catalyzed by SIRT1 and their functions.Through several TET2 immune precipitation assays in MDSL cells combined with mass spectrometry protein assay,we detected the stable acetylated residues K1468,K1472,K1473 and K1478.However,SIRT1 overexpression significantly reduces the acetylation level of the four lysine sites,indicating the acetylation level of the four sites regulated by SIRT1.Because TET2 is a DNA demethylase,which catalyze the 5-methylcytosine(5mc)into 5-hydroxymethyl cytosine(5hmc),thus its function can be detected by measuring the alteration of the DNA 5hmc level.To detect whether the acetylation level of these four lysine sites affects TET2 function,we engineered the corresponding mutants mimic loss of acetylation,then transduced into MDSL-TET2KD cells.Enzyme-linked immunosorbent assay was used to detect the 5hmc changes.The results showed that K1472,K1473 and K1478 sites affected 5hmc level,indicating related with TET2 function.So how does the acetylation in TET2 protein affect its DNA demethylation function?According to relevant literatures,we supposed that it may be associated with DNA binding ability.Therefore,we further carried out EMSA assay.The results showed that TET2 acetylation reduction increases its DNA binding capacity.Finally,the rescue experiment suggested that the function of SIRT1 overexpression or activation in inhibiting MDS tumor proliferation was mainly through TET2 de-acetylation.Acetylation sites of K1472,K1473 and K1478 exert the main functions.3.SIRT1 agonist SRT1720 effectively inhibits MDS cell proliferationFirstly,the result of MDSL cells engrafted mouse model showed that SRT1720 gavage significantly decreases the engraftment and prolong the overall survival time.Secondly,the cloning formation assay showed that SRT1720 inhibited the in vitro cloning formation and in vivo engraftment ability of primary MDS cells in a dose-dependent manner.Finally,SRT1720 long-term gastric administration significantly alleviated cytopenia and bone marrow dysplasia,decrease the proportion of donor cells in peripheral blood and bone marrow in the NHD13 MDS transgenic mouse model.The results of the secondary transplantation were similar.All of these showed the therapeutic efficiency of SRT1720 in inhibiting MDS cells proliferation.Research conclusionTogether,this study shows that SIRT1 is low expressed in MDS and that SIRT1 low expression promotes MDS cell proliferation by increasing TET2 acetylation.High expression or SIRT1 agonist SRT1720 significantly decreases TET2 acetylation levels,inhibits its DNA binding ability,thus weakens the function of TET2 in DNA demethylation,leading to MDS cell proliferation inhibition both in vivo and in vitro.