Study On The Regulation Of THAP11 On The Function And Development Of Hematopoietic Stem Cells

THAP11 is the newest member of the THAP?Thanatos-associated protein?family.The Thap11 gene in mice is usually called Ronin.Ronin was first found in the early stage of mouse embryonic development,which is necessary to maintain stem cell pluripotency in vivo and in vitro.Existing studies have shown that THAP11 regulates cell proliferation,differentiation,apoptosis and mitochondrial function mainly through transcription factor interaction networks.It is mainly reflected in the process of inhibiting the growth of tumor cells,regulating the dryness of embryonic stem cells and regulating the proliferation and differentiation of pluripotent stem cells.However,the regulatory effect of THAP11 on hematopoietic stem cells has not been reported.Previous studies in our laboratory have found that THAP11 regulates the balance of erythroid/megakaryocyte differentiation in erythroleukemia cell line?K562?,which suggests that THAP11 may play an important role in hematopoietic regulation.In this study,human primary cord blood CD34⁺cells and conditional knockout mouse models were used to systematically analyze the effects of THAP11 on HSC function and hematopoietic system development.In this study,based on the RNAi lentiviral system,the primary blood cells of human blood cord blood?CB?CD34⁺knockdown THAP11,and the effect of THAP11 on HSC in vivo reconstruction,growth,proliferation,cycle and apoptosis in vitro were detected.The results showed that three months after transplantation of CB CD34⁺cells interfering with THAP11 expression into immunodeficient mice irradiated with lethal dose,the reconstruction rate of human GFP⁺cells in THAP11 interference group was significantly lower than that in control group.The results of colony culture in vitro showed that the number of colonies formed by CB CD34⁺cells decreased significantly after THAP11 interference,which was related to the interference efficiency.In the in vitro amplification culture system,after THAP11 interference,the proliferation of CB CD34⁺cells decreased in the process of culture in vitro,the proportion of G0/G1 phase cells of CB CD34⁺cells increased,and the apoptosis of THAP11 interference group increased after the withdrawal of all cytokines.In the megakaryocyte induction culture system,the differentiation of CB CD34⁺into megakaryocyte line decreased after THAP11 interference,and the erythroid differentiation of CD34⁺cells decreased after interference with THAP11 in erythroid induction culture system.These results suggest that THAP11 plays an important role in regulating the function of human HSC,but the regulatory mechanism needs to be further studied.On the basis of previous laboratory studies,Ronin gene conditional knockout?Conditional Knockout,CKO?mice were constructed based on LoxP-Cre system.the proportion of offspring genotypes produced by mating between Ronin^{flo x/flo x}?flanked by loxP?mice and Ronin^flox/+Vav-iCre⁺mice was calculated.The results showed that the actual birth rate of Ronin^{flo x/flo x}Vav-iCre⁺mice did not conform to Mendel's law,and only one mouse was born and died within one week,indicating that the specific knockout of Ronin in the hematopoietic system may lead to the inability of embryos to survive.Mouse embryos at 14.5dpc?days post coitum?,15.5dpc and 18.5dpc were dissected to observe the morphological phenotype of Ronin CKO mice.The results showed that the embryo of Ronin CKO mice was pale,and the three main hematopoietic organs of yolk sac,placenta and fetal liver were obviously white.With the passage of time,the morphological phenotype increased,the fetal liver became smaller and the number of cells decreased,the red blood cell denucleation in circulating blood was disturbed,and the embryo showed severe anemia phenotype.CD71 and TER119 were used to label red blood cells at different developmental stages to further analyze the cell composition of mouse fetal liver at 14.5dpc and 15.5dpc.The results showed that the erythroid development was blocked in the stage of CD71^hiTer119⁺cells,and the number of Ter119⁺cells in the whole fetal liver was seriously decreased.Further analysis of Ter119⁺cells showed that arrest occurred in the process of cell progression from large to small.Lin^-c^-Kit⁺Sca1^-labeled hematopoietic progenitor cell?Hematopoietic progenitor cell,HPC?and Lin^-c-Kit⁺Sca1⁺labeled hematopoietic stem cell?Haematopoietic stem cell,HSC?,were used to analyze the composition of hematopoietic stem progenitor cells in fetal liver.The results showed that the number of Lin^-c-Kit⁺Sca1⁺cells in the fetal liver of Ronin CKO mice increased significantly at 13.5 days,and there was no significant difference in cell number when the embryos developed to 15.5 days.At 13.5days,there was no significant difference in the number of Lin^-c-Kit⁺Sca1^-cells,and the number of it was significantly reduced at 15.5 days of development.The results of in vitro colony formation experiments showed that the colony forming ability of 13.5 dpc fetal liver cells decreased significantly after Ronin deletion.Apoptosis was significantly increased in fetal liver of 13.5dpc,14.5dpc,and 15.5dpc Ronin CKO mice.The establishment of Ronin CKO mouse model confirmed that Ronin played an important role in the regulation of hematopoietic system,resulting in severe ischemia of Ronin CKO mouse embryos and unable to survive normally.The preliminary results of embryonic phenotype showed that the regulation of Ronin in the early hematopoietic development may play an important role in two nodes.On the one hand,the conditional deletion of Ronin blocks the terminal differentiation of erythroid during embryonic erythroid development.It is difficult to produce mature red blood cells.It leads to severe anemia in embryos,which may be related to mitochondrial clearance during erythrocyte development and maturation.On the other hand,the conditional deletion of Ronin affects the function of hematopoietic stem cells,which seems to be consistent with the conclusion in human CB CD34⁺.It eventually leads to the development of the whole hematopoietic system of the embryo.In conclusion,this study explored the regulatory effect of THAP11 on hematopoietic stem cell function in human CB CD34⁺primary cell model and Ronin CKO mouse model interfering with THAP11,and confirmed that THAP11 played an important role in hematopoietic system.It provides a reliable clue and an animal model for the follow-up study of the mechanism of THAP11 regulating hematopoietic stem cell function.