| Backgro?nd:Hematopoietic differentiation is the process of hematopoietic stem cells after directional differentiation and eventually become a variety of mature cells,among which,erythroid differentiation is an important part.The differentiation and maturation disorder is the characteristic of leukemia once red line development obstacle,block the formation of reticulocyte,terminal differentiation is inhibited,then peripheral blood have nucleated red cell increase,gradually replaced the normal hematopoiesis,and invaded the liver,lung and other organs and systems,so that the patient appeared the characteristics of pernicious anemia,bleeding,infection and infiltration,and eventually died.Leukemia is a kind of malignant clonal disease of hematopoietic stem cells with the characteristics of uncontrolled proliferation,apoptosis block,hematopoietic function inhibition and differentiation disorder.Chronic myeloid leukemia(chronic myelocytic leukemia,CML)accounts for about 20% of all adult leukaemia and generally has BCR-ABL characteristic Ph chromosomes.K562 is a cell line established from the pleural effusion of CML female patients with stem cell characteristics.it is a cell line of human red and white blood disease.it is more behavioral like early undifferentiated multi-potential hematopoietic progenitor cells,it can be used as an ideal model to induce differentiation after a variety of chemical inducers in vitro.MiRNAs abnormal expression is related to the occurrence and development of many diseases,especially cancer.recent studies have shown that miRNAs is closelyrelated to hematopoietic cell proliferation,differentiation,blood system diseases,and plays an important role in the process of erythroid differentiation,proliferation and erythrocyte maturation.miR-429 is one of the members of the miR-200 family,and its abnormal expression is closely related to the occurrence,development,metastasis,apoptosis,drug resistance and so on.Signal junction protein CRKL(v-crk sarcoma virus ct10 oncogene homologue(avian)-like)is a member of the CRK family and contains one SH2 and two SH3 domains.MiRNAs is involved in regulating the growth and development of various tumor cells,especially in the proliferation,differentiation,maturation and apoptosis of hematopoietic cells related to diseases of the blood system.CRKL can recruit signal pathway molecules to participate in cell signal transduction process by SH2?SH3 domain,and then affect cell differentiation,proliferation,adhesion,migration,invasion,apoptosis and so on,which play the role of signal switch.The results showed that CRKL expression increased in bone marrow samples of patients with primary CML and promoted erythroid differentiation of K562 cells after knockdown.At the same time,miR-429 directly targeted CRKL-3'-UTR negatively regulated expression.There was also a negative correlation between the two expression levels in HCC tissue samples.We speculate that miR-429 may be associated with erythroid differentiation,the relationship between miR-429 and leukemia and erythroid differentiation has not been reported.In this paper,we will study the effect and preliminary mechanism of miR-429 on erythroid differentiation of K562 cells by upregulating the expression in the cells.Objective:1.To detect the expression level and correlation analysis of the expression levels of miR-429 and CRKL in bone marrow samples of CML patients.2.To verify the expression level and correlation analysis of miR-429,CRKL in K562 cell erythroid differentiation.3.To specificy the effect of miR-429 overexpression on K562 cell erythroid differentiation.4.To have a preliminary study on the mechanism of miR-429 regulating erythroiddifferentiation.Methods:1.qRT-PCR detection of miR-429 ? CRKL expression in CML primary patient samples,primary-complete remission(CR)paired samples,and analyzation of their correlation.2.Benzidine Dyeing detected the effects of different concentrations of Hemin on red line differentiation in K562 cells.3.MTT assay detected the effect of different concentrations hemin on proliferation of K562 cells.4.q RT-PCR detected the expression level of expression levels of erythroid differentiation markers GPA and globin following Hemin-induced erythroid differentiation in K562 cells.5.q RT-PCR and Western blotting(WB)levels detect the expression of miR-429,CRKL during Hemin-induced erythroid differentiation.6.Synthetic miR-429 mimic and their NC,were transiently transfected into K562 cells to up-regulate miR-429 expression.7.Hemin induced erythrogenic differentiation in K562 cells,benzidine staining detect the effect of changes in the expression level of miR-429 on the expression of hemoglobin in K562 cells8.Hemin induce erythroid differentiation in K562 cells,q RT-PCR detect the effect of changes in expression level on molecular ?-globin,?-globin and GPA expression of erythroid differentiation markers in K562 cells.Results:1.The expression of miR-429 was lower in bone marrow samples of CML primary patients CRKL higher in bone marrow samples of CML primary patients compared with normal samples.2.The expression of primary-CR and recurrent-CR pairs of CML showed an increasing trend of miR-429 after complete remission and a decreasing trend of CRKL expression.3.miR-429 and CRKL expression levels were negatively correlated.4.The proportion of benzidine staining positive cells increased with the increase of Hemin concentration and the time of induction,but the excessive concentration caused the death of a large number of cells in a dose-dependent and time-dependent manner after Hemin induced erythroid differentiation of K562 cells.5.Hemin K562 cell erythroid differentiation induced by induction increases with the Hemin concentration,increases the ability to inhibit cell proliferation in K562 cells,but the excessive concentration caused the death of a large number of cells in a dose-dependent and time-dependent manner after Hemin induced erythroid differentiation of K562 cells.6.The ?-globin,?-globin and GPA expression levels of erythroid differentiation markers increased after 60 ?M Hemin induced erythroid differentiation in K562 cells,indicating the successful induction of erythroid differentiation.7.The expression levels of miR-429 and CRKL increased and decreased during Hemin-induced erythroid differentiation,and their expression levels were negatively correlated.8.The miR-429 mimic and its NC were transiently transfected into K562 cells,which were miR-429 successfully overexpressed.9.miR-429 overexpression increases the proportion of benzidine-stained positive cells and promotes hemoglobin expression;it also promotes ?-globin,?-globin and GPA expression.Conclusion:1.The expression of miR-429 was lower in CML patients' bone marrow samples,higher in CR patients,and CRKL was higher in CML patients' bone marrow samples and lower in CR patients,that indicates a significant negative correlation between them in clinical level.2.The expression levels of miR-429 and CRKL increased and decreased in the process of K562 cell erythroid differentiation,which shows a significant negative correlation with the expression of them at cell level.3.miR-429 overexpression promotes K562 erythroid differentiation.4.miR-429 may regulate erythroid differentiation by targeting CRKL. |
PDF Full Text Request