| Background:Erythroid differentiation refers to the process that hematopoietic stem cells develop into mature red blood cells after multiple growth and differentiation stages,such as pluripotent hematopoietic stem cells,erythroid progenitor cells,erythroid progenitor cells,reticulocytes and so on.it is an important way for the body to produce mature red blood cells.Once the development of erythroid cells stagnates at a certain stage and cannot finally differentiate into mature red blood cells,it will lead to a variety of diseases,of which leukemia is the most serious and common one.Chronic myeloid leukemia(CML)is a malignant clonal disease originating from bone marrow pluripotent hematopoietic stem cells,accounting for about 20% of all adult leukemia.The disorder of differentiation and maturation is considered to be the commonness of leukemic cells.Therefore,inducing leukemic cells to differentiate and break through the barrier of differentiation and maturation has become the focus of medical basic research and clinical transformation.K562 cell line is a cell line isolated from CML patients.It has the characteristics of stem cells and is closer to the multi-potential hematopoietic progenitor cells in the early undifferentiated stage.It is an ideal model to induce cell differentiation.ETV6,also known as TEL,located in the short arm region(12p13)of human chromosome 12,is a member of the E26 transformation-specific(ETS)family.It encodes a protein with 452 amino acids and has two unique domains: the N-terminal HLH domain and the C-terminal ETS domain.Because its domain can combine with other molecules to form different chimeric products,such as ETV6-RUNX1,ETV6-PDGFRβ and ETV6-ABL fusion genes,it is involved in the formation of leukemia.In addition,as a transcriptional suppressor,the main role of ETV6 is to inhibit the expression of a variety of target genes,many of which are highly regulated in the process of hematopoiesis,indicating that there is a close relationship between ETV6 and erythroid differentiation of leukemic cells.In the previous study,it was found that the signal junction protein CRKL,which is closely related to ETV6 and has a direct binding and regulatory relationship,is closely related to the erythroid differentiation of K562 cells.Combined with the important role of ETV6 in the occurrence and development of a variety of hematological malignant tumors,we speculate that there is a certain relationship between ETV6 and erythroid differentiation of K562 cells.Objective:1.To detect the expression level of ETV6 in CML patients and analyze the correlation with CML.2.To determine the effect of ETV6 on erythroid differentiation of K562 cells3.To explore the molecular mechanism of erythroid differentiation of K562 cells regulated by ETV6.Methods:1.The expression of ETV6 in peripheral blood mononuclear cells of 13 patients with CML,5 patients with complete remission of CML and 7 normal donors was detected by q RT-PCR.2.After K562 cells were induced by hemin for 0,12,24,36 and 48 h,the positive rate of erythroid differentiation of K562 was detected by benzidine staining and the expression of ETV6 was detected by Western blot.3.Benzidine staining was used to detect the effect of ETV6 expression on erythroid differentiation of K562.4.The effect of ETV6 expression on the expression of erythroid differentiation-related molecules in K562 was detected by q RT-PCR.5.CCK-8 assay was used to detect the effect of ETV6 expression on the proliferation of K562 cells.6.Flow cytometry was used to detect the effect of ETV6 expression on the expression of erythroid differentiation marker molecules GPA and CD71 in K562.7.The effect of ETV6 expression on Raf/MEK/ERK pathway molecules in K562 was detected by Western blot.8.PD98059 inhibited Raf/MEK/ERK pathway,Western blot to detect the expression of ERK and p-ERK in K562 cells,benzidine staining was used to detect the positive rate of erythroid differentiation of K562 cells,and q RT-PCR was used to detect the expression of erythroid differentiation-related molecules in K562 cells.Results:1.Compared with the peripheral blood mononuclear cells of normal donors,the overall expression level of ETV6 in CML patients was 137.3% higher.After complete remission,the expression level of ETV6 in patients with complete remission was 54.5%lower than that in patients with CML,and there was no difference in the expression level of ETV6 between normal subjects and normal subjects.2.When K562 was induced by hemin,the expression of ETV6 decreased continuously with the increase of induction time.3.After knocking down ETV6,the positive rate of erythroid differentiation in K562 cells increased by 26.9%(P = 0.0017),and the expression levels of erythroid differentiation-related molecules α-globin,γ-globin,ε-globin,GPA and HBA increased by 44.8%(P < 0.0001),38.5%(P < 0.0135),23.8%(P < 0.0183)and 112.3%(P <0.0142),respectively.CCK-8 assay showed that the cell proliferation rate decreased by6.6% at 24 h,20.6% at 48 h and 22.0% at 72 h(P < 0.0001).The ability of cell proliferation is decreased.4.After overexpression of ETV6,the positive rate of erythroid differentiation in K562 cells decreased by 25.7%(P = 0.0016),and the expression levels of erythroid differentiation-related molecules α-globin,γ-globin,ε-globin,GPA and HBA decreased by 49.5%(P < 0.0001),33.5%(P = 0.0018),30.9%(P = 0.0051),25.1%(P =0.0123)and 25.6%(P = 0.0086),respectively.The cell proliferation rate increased by13.0% at 24 h(P = 0.0003),24.2% at 48 h(P < 0.0001)and 15.9% at 72 h(P =0.0004),measured by CCK-8 method.5.After knocking down ETV6,p-Raf,p-MEK and p-ERK increased by 18.8%,61.5% and 37.9%,respectively,while there was no significant change in the expression of Ras,Raf,MEK and ERK prototype molecules.After overexpression of ETV6,p-Raf,p-MEK and p-ERK decreased by 43.0%(P = 0.0149),36.6%(P = 0.0066)and 45.6%(P = 0.0382).There was no significant change in the expression of Ras,Raf,MEK and ERK prototype molecules.6.The blocking of ERK pathway weakens the promoting effect of down-regulation of ETV6 on the positive rate of erythroid differentiation in K562 cells,while the blocking of ERK pathway weakens the down-regulation of ETV6 on the expression of erythroid differentiation-related molecules.Conclusion:1.ETV6 is highly expressed in patients with primary CML,but the level of ETV6 is significantly decreased in patients with CML after complete remission,indicating that the increase of ETV6 is related to the pathogenesis of CML.2.ETV6 knockdown promotes erythroid differentiation of K562 cells,and overexpression of ETV6 inhibits erythroid differentiation of K562 cells.3.ETV6 knockdown activates Raf/MEK/ERK pathway activity.ETV6 overexpression inhibited RAF /MEK/ERK pathway activity. |
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