Study On The Mechanism Of ANTXR1 Regulating γ-globin Expression Through Wnt/β-catenin Signaling Pathway And Its Effect On Erythroid Differentiatio

It was shown that reactivation ofγ-globin gene(HBG1 and HBG2)expression inβ-thalassemia patients has been shown to be an effective therapy forβ-thalassemia.The regulatory mechanism ofγ-globin expression is still unknown.Patients withβ-thalassemia have erythrocytes that do not produce enough normal hemoglobin,resulting in abnormal blood cell changes and the destruction of large numbers of abnormal erythrocytes in the bone marrow.Thus,abnormal hematopoiesis leads to a shorter lifespan,also known as"ineffective hematopoiesis".The regulation of erythroid differentiation is a multi-step and multi-layered complex process that is influenced by a number of factors,including transcription factors and non-coding RNAs.Our group discovered that silencing of the anthrax toxin receptor 1(ANTXR1)gene in K562 cells could promoteγ-globin expression,but the molecular mechanism by which ANTXR1regulatesγ-globin expression remains unknown.Therefore,in this study,for the first time,the molecular mechanism of ANTXR1 regulation ofγ-globin expression is investigated from a cytological point of view.The effects of ANTXR1 on the proliferation and differentiation of hematopoietic stem cells during the development of erythroid differentiation have also been investigated.The present data will provide a theoretical basis for further elucidation of the mechanism ofγ-globin expression regulation.Part I.Effect of ANTXR1 onγ-globin expressionObjective:To understand the differential expression of ANTXR1,γ-globin,andβ-catenin in the Wnt signaling pathway during erythroid differentiation of K562 cells,cord blood and adult peripheral blood CD34~+cells,and erythroid progenitor cells(human umbilical cord blood-derived erythroid progenitor,HUDEP-2).To investigate the relationship between ANTXR1 andβ-catenin gene expression andγ-globin expression during erythroid differentiation.To investigate the effect of ANTXR1 onγ-globin expression,lentiviruses overexpressing and disrupting the ANTXR1 gene were infected with the above cells.Methods:1.CD34~+cells from cord blood and adult peripheral blood were cultured and subjected to erythroid differentiation.ANTXR1,γ-globin,andβ-catenin proteins were detected by Western blot during erythroid differentiation of the above cells.2.Overexpression and disruption of ANTXR1 gene lentivirus infection of the above cells.RT-q PCR and Western blot were used to detectγ-globin expression during red lineage differentiation.Results:1.The expression levels of ANTXR1,β-catenin,andγ-globin were low in cord blood CD34~+cells during early red lineage differentiation but gradually increased as red lineage differentiation progressed.Compared with day 14 of red lineage differentiation,the expression of ANTXR1 andβ-catenin protein tended to decrease on days 15 and 16,whereas the expression ofγ-globin remained high.Similarly,the kinetics of erythroid differentiation of adult peripheral blood CD34~+cells,HUDEP-2 cells,expression of ANTXR1,β-catenin,andγ-globin mirrored that of cord blood CD34~+cells.ANTXR1,β-catenin,andγ-globin may have a regulatory relationship during this time.2.Compared with the control group,overexpression of the ANTXR1 gene suppressedγ-globin expression in the m RNA and protein levels of K562 cells,cord blood CD34~+cells,and red lineage differentiation.In contrast,ANTXR1 gene interference increasedγ-globin expression at the m RNA and protein levels in K562 cells,cord blood CD34~+cells,and HUDEP-2 cells during red lineage differentiation compared with controls.It was suggested that ANTXR1 has a negative regulatory effect on theγ-globin.Conclusion:ANTXR1 andγ-globin andβ-catenin have a regulatory relationship,and ANTXR1negatively regulatesγ-globin in the late phase of the red lineage differentiation process.Part II Mechanism of ANTXR1 regulation ofγ-globin expression through Wnt/β-catenin signaling pathwayObjective:To investigate the mechanism by which ANTXR1 regulatesγ-globin expression via the Wnt/β-catenin pathway,Wnt/β-catenin pathway activity,and major gene expression changes were observed in K562 cells after overexpression and disruption of the ANTXR1 gene.Methods:1.An immunofluorescence assay was used to detect the co-localization of ANTXR1with low-density lipoprotein receptor protein 6(LDL receptor protein,LRP6).To detect the interaction between ANTXR1 and LRP6,the immunoprecipitation(Co-IP)technique was used.2.Immunofluorescence and nucleoplasma separation assays were used to detect the localization ofβ-catenin in the Wnt/β-catenin pathway after overexpression and interference of the ANTXR1 gene.RT-q PCR and Western blot were used to detect m RNA and protein expression of LRP-6,β-catenin,c-Jun,and cyclin D1 in the Wnt/β-catenin pathway.3.The TOP/FOP flash luciferase reporter system was used to examine the altered activity of the Wnt/β-catenin pathway after overexpression and interference of the ANTXR1 gene.4.Before performing a reversion assay,we added 2.5,5,and 10μmol/L of the Wnt signaling pathway inhibitor XAV939 and 5,20,and 40 m M of the activator Li Cl to K562 cells stably transfected with overexpressed or interference ANTXR1 genes,respectively,to observe the expression of theγ-globin and Wnt pathway.5.By Ch IP-q PCR,changes in the expression of SOX6,EIF2AK,BGLT3,and ZBTB7A were detected after overexpression of ANTXR1.6.A K562 cell line overexpressing the c-Jun gene was created,and protein expression of theγ-globin suppressor gene SOX6 was detected by Western blot.Results:1.Immunofluorescence showed that ANTXR1 co-localizes with LRP6 on the K562 cell membrane and that ANTXR1 and LRP6 interact.2.Overexpression of the ANTXR1 gene activated the Wnt/β-catenin signaling pathway and promoted the entry ofβ-catenin into the nucleus,compared with the control group.Therefore,cytosolicβ-catenin protein expression increased more than cytoplasmicβ-catenin protein expression.In contrast,ANTXR1 gene interference inhibited the Wnt/β-catenin signaling pathway so thatβ-catenin left the nucleus and cytoplasmicβ-catenin protein expression increased more than nuclearβ-catenin protein expression.3.Different XAV939 concentrations significantly reversed the inhibitory effect onγ-globin expression after ANTXR1 gene overexpression.After the impairment of ANTXR1gene,different concentrations of Li Cl significantly reversed the promoting effect onγ-globin expression.4.The four genes that inhibitedγ-globin expression(SOX6,EIF2AK,BGLT3,and ZBTB7A)had c-Jun binding sites in their transcriptional regulatory regions,which were discovered by reviewing the Ch IP-seq public database.The ability to bind to c-Jun was greatly enhanced.The transcriptional regulatory regions of the remaining genes showed no significant changes in their ability to bind to c-Jun.5.overexpression of the c-Jun gene increased the expression of theγ-globin repressor gene SOX6 protein.Conclusion:ANTXR1 can inhibitγ-globin expression by activating the Wnt/β-catenin signaling pathway,promotingβ-catenin entry into the nucleus,and initiating binding of the downstream transcription factor c-Jun to the transcriptional regulatory region of the SOX6 gene encoding theγ-globin repressor.Part III Effect of ANTXR1 on proliferation and differentiation of hematopoietic stem cells in vitroObjective:Overexpressing and disrupting ANTXR1 to see how it affects the proliferation and differentiation of hematopoietic stem cells in vitro.Methods:1.CCK8 and flow cytometry were used to determine the effects of ANTXR1 gene overexpression and interference on hematopoietic stem cell proliferation and differentiation during red lineage differentiation.2.Benzidine and Wright-Giemsa staining were used to assess the effects of ANTXR1gene overexpression and interference on hematopoietic stem cell proliferation and differentiation during red lineage differentiation.3.The effects of ANTXR1 gene overexpression and interference on the transcription factors GATA1 and ALAS2 and the protein levels of GSK3β,β-catenin,and p-β-catenin of the Wnt/β-catenin pathway in hematopoietic stem cells during red lineage differentiation were examined by RT-q PCR and Western blot.4.Reversion assay was performed with different concentrations of XAV939 and Li Cl.The aim is to investigate the effects of ANTXR1 gene overexpression and interference on the proliferation and differentiation of hematopoietic stem cells during the red lineage differentiation process.Results:1.Overexpression of the ANTXR1 gene promoted the proliferation of hematopoietic stem cells while inhibiting differentiation and apoptosis.In addition,interference of the ANTXR1 gene inhibited proliferation and promoted differentiation and apoptosis in hematopoietic stem cells.2.ANTXR1 gene overexpression and interference inhibited and promoted erythroid transcription factors GATA1 and ALAS2 m RNA and protein expression,respectively.3.Overexpression of the ANTXR1 gene increasedβ-catenin protein expression,whereas GSK3βand p-β-catenin protein expression decreased.Disruption of the ANTXR1 gene inhibited the expression ofβ-catenin protein,whereas the expression of GSK3βand p-β-catenin protein increased.4.Different concentrations of XAV939 significantly inhibited the promoting effect of ANTXR1 gene overexpression on hematopoietic stem cell proliferation,whereas Li Cl significantly reversed the inhibitory effect of ANTXR1 gene interference on hematopoietic stem cell proliferation.Conclusion:Overexpression of the ANTXR1 gene promotes the proliferation of hematopoietic stem cells while inhibiting cell differentiation and apoptosis.Furthermore,disruption of the ANTXR1 gene inhibits the proliferation of hematopoietic stem cells while promoting cell differentiation and apoptosis.The hypothesis is that ANTXR1 regulates the proliferation and differentiation of hematopoietic stem cells.