The Mechanisms Of TET2 On Regulating Human Erythroid Progenitors

Research background and purpose:Erythropoiesis is the formation process of functional mature red blood cells from hematopoietic stem cells through important biological events such as cell self-renewal,proliferation,differentiation and enucleation in the hematopoietic microenvironment.Anemia caused by erythropoiesis or ineffective hematopoiesis is an important clinical manifestation of many human hematological diseases,including myelodysplastic syndromes(MDSs).MDSs are clonal hematopoietic stem cell disorders.And the main symptoms are defects in bone marrow hematopoietic function and apoptosis of intramedullary cells.However,the most frequently mutated gene in patients with MDSs is Ten-Eleven-Translocation 2(TET2),which functions to convert5-methylcytosine(5-mC)to 5-hydroxymethylcyosine(5-hmC),which participates in the process of DNA demethylation,and it affects target gene transcription and expression.At present,the molecular mechanism of erythroid abnormalities in MDSs patients with TET2 mutation remains unclear.It was reported that TET2 knockdown caused clonal proliferation of c-Kit and AXL-mediated erythroid progenitor cells,resulting in dysfunctional Marker-CFU-E(M-CFU-E)cells.We further identified that M-CFU-E cells abnormally proliferated and caused a large amount of apoptosis.Therefore,We mainly studied TET2 knockdown in the erythroid culture system in vitro and hematological tumor cell line,in order to reveal the molecular mechanism of apoptosis of erythroid progenitor cells of TET2 in regulating.This study provides a new perspective for the mechanism of apoptosis associated with MDSs disease in TET2 mutation,and provides a new scientific basis for the treatment of MDSs diseases.Methods:Firstly,we used CD34~+magnetic beads to isolate hematopoietic stem cells with more than 95%purity in mononuclear cells of cord blood,CD34~+cells were transfected with lentivirus carrying TET2-shRNA and screened with puromycin.qRT-PCR was performed to examined the efficiency of TET2 knockdown,and TET2knockdown cells were successfully obtained.In the erythroid culture system in vitro,the erythroid progenitor cells were sorted by flow cytometry,and terminal differentiation and apoptosis were tested by flow cytometry,morphological observation was assessed by cytospins.Secondly,in order to identify the relevant apoptotic molecules,we sorted out Luciferase CFU-E,TET2-CFU-E and M-CFU-E cells for transcriptome sequencing(RNA-seq).Bioinformatics analysis was used to test differential genes and apoptosis-related signaling pathways after TET2 knockdown.To verify the differential genes in expression,qRT-PCR and flow cytometry were used to examined the expression of apoptosis-related molecules(FAS,Fas Cell Surface Death Receptor)in TET2 knockdown cells.To further investigate the molecular mechanism by up-regulated expression of FAS induced by TET2 knockdown,we detected and analyzed the differences in 5-mC and 5-hmC of key CCGG sites in the FAS promoter region by glycosylation and restriction endonuclease treatment.Meanwhile,the caspase 3 specific inhibitor(Z-DEVD-FMK)in the downstream pathway of FAS were used to further verify the apoptotic pathway.Moreover,we also selected hematopoietic stem cells from the bone marrow of healthy people and MDSs patients with TET2 mutation,and we checked apoptosis and the expression of FAS after inducing differentiated into erythroid cells in vitro.Finally,because TET2 mutation was involved in the development of various hematological tumors.Therefore,we knocked down the expression of TET2 in erythroleukemia cell line K562 cells,and we examined proliferation and apoptosis.And FAS was overexpressed in K562 cells to detect its effects on proliferation and apoptosis.Results:Firstly,in order to explore the function of TET2 during erythroid progenitor cell development,we initially verified TET2 knockdown efficiency of CD34~+cells infected with TET2-shRNA.We found that the expression level of TET2 in the experimental group was approximately 40%of the expression level of TET2 in the control group.On this basis,TET2-CFU-E was sorted and cultured in erythroid development system in vitro.We found that terminal differentiation was blocked with TET2 knockdown.To investigate associated phenotypes of cells with blocked differentiation,the M-CFU-E cells was sorted and cultured in erythroid culture system in vitro,and we found that the apoptosis was significantly increased.Secondly,in order to clarify its related apoptotic molecules,we performed RNA-seq analysis of Luciferase CFU-E and M-CFU-E.Bioinformations analysis revealed that about 400 genes were differential expressed,included 25 genes related to apoptosis-related signaling pathways,and 16 of which were up-regulated,such as FAS,NCKAP1 and TNFRSF10B and etc.Our qRT-PCR and flow cytometry analysis revealed that compared with Luciferase CFU-E,the expression of FAS in TET2-CFU-E cells was unchanged,while the expression in M-CFU-E cells was up-regulated.In addition,in order to investigate the mechanism of FAS up-regulation caused by TET2 knockdown.Analysis of 5-mC and 5-hmC in the FAS promoter region key CCGG sites(-305,-251,-136,-90)of the above three group cells showed that compared with Luciferase CFU-E,the 5-mC and 5-hmC of FAS key CCGG sites in TET2-CFU-E and M-CFU-E cells did not change.The expression of FAS might not be direct regulated by TET2,and there were other target genes between TET2 and FAS to regulate gene expressed.Moreover,to further investigate whether TET2deficiency causes apoptosis by regulating Caspase 3,rescue experiment showed that compared with Luciferase CFU-E,we found that Caspase 3 specific inhibitor(Z-DEVD-FMK)failed to rescue apoptosis in M-CFU-E cells,indicating that apoptosis induced by early erythroid progenitor cells after TET2 knockdown did not depend on the apoptotic pathway of Caspase 3.Importantly,we found that compared with healthy people,hematopoietic stem cells of bone marrow cells of MDSs patients with TET2 mutation significantly increased apoptosis on the day11 and day13 and the expression of FAS was up-regulated from day11 to day17.Finally,the conversion of leukemia to high risk is an important feature of MDSs disease,and apoptosis plays an important role in the occurrence and evolution of MDSs.To investigate the role of TET2-regulated apoptosis in the evolution of MDSs,compared the expression levels of TET2 and FAS in healthy human bone marrow cells and K562 cells,the results showed that the expression of TET2 in K562 cells was half of the healthy people's bone marrow cells,and FAS was not expressed in K562 cells.To further clarify the function of TET2 in K562 cells,we obtained K562cells with TET2 knockdown.Subsequently,we induced K562 cells to differentiate into erythroid differentiation by Hemin.We found that compared with the control group,TET2 knockdown promoted cell proliferation and did not affect apoptosis,and the expression of FAS did not change.Moreover,overexpression of FAS in K562cells revealed that high levels of FAS promoted apoptosis and inhibited proliferation.This result suggests that the expressed level of FAS may be an important regulator of the evolution of TET2 mutant MDSs disease,and FAS and its downstream signaling pathway may serve as potential drug targets for the treatment of hematological tumors.Conclusion:In summary,we explored the important function of TET2 in the erythroid progenitor cell stage:TET2 knockdown produced dysfunctional M-CFU-E cells,which was blocked by differentiation,increased apoptosis and up-regulated expression ofFAS.TET2 knockdown did not cause change in methylation and demethylation of key sites in FAS promoter region.Therefore,TET2 might indirectly regulate the expression of FAS.Moreover,apoptosis induced by TET2 knockdown,it was independent of the Caspase 3 pathway.Meanwhile,bone marrow cells of MDSs patients with TET2 mutations were also accompanied by a large number of apoptosis,and the expression of FAS was significantly up-regulated.Furthmore,TET2knockdown could also promote the proliferation of K562 cells,but did not affect apoptosis.However,Overexpression of FAS could promote a large number of apoptosis in K562 cells.Therefore,the expression level of FAS might be an important marker for the transformation of MDSs disease caused by TET2 mutation into leukemia.Our findings provide a new perspective for the disease-related apoptosis mechanism of TET2 mutant MDSs and scientific basis for the treatment of MDSs diseases.