| High altitude polycythemia(HAPC)is a common high altitude chronic disease that can lead to an increase erythrocytosis,which seriously affects the health of high altitude residents.Although chronic hypoxia is the main cause of HAPC,the related molecular mechanisms responsible for its development remain largely unclear yet.GATA-1,which is a transcription regulator of erythroid differentiation,is crucial to erythroid differentiation.GATA-1 regulates miR-451 a that is a erythroid specific micro RNA and participates in the process of red line differentiation.The expression of GATA-1 is regulated by hypoxia,but it is unknown that GATA-1 regulates miR-451 a is involved in erythrocytosis under hypoxia.In this study,K562 cells and CD34~+ hematopoietic stem/progenitor cells of HAPC patients were used as subjects,from in vitro cell model studies to clinical samples,to explore the correlation between GATA-1 and miR-451 a in erythroid differentiation under hypoxia.This study consists of two parts as follows:Part ? Expression Relevance of GATA-1 and miR-451 a on Erythroid Differentiation of K562 cells under HypoxiaObjective:To investigate the expression relevance of GATA-1 and miR-451a(micro RNA-451 a,miR-451a)on erythroid differentiation of K562 cells under hypoxia.Methods : K562 cells were divided into the normoxia group and the hypoxia group(1% O2),and 40 ?M hemin was used to induce K562 cell differentiation for 48 h and 72 h,respectively.The expression of two erythroid differentiation indicators includes ?-globin and CD235 a were detected to verify whether the K562 cell erythroid differentiation model was replicated successfully.After that,the protein expression of GATA-1 was detected by Western blot,and the RNA expression of GATA-1 m RNA and miR-451 a were detected by q RT-PCR and correlation analysis was performed.The erythroid differentiation indicators of K562 cells were dectected in overexpression,inhibition or negative control groups of GATA-1.Meanwhile,the morphological changes of cells in the above groups were analyzed by using Wright-Giemsa staining method,to clarify the erythroid differentiation of K562 cells after GATA-1 overexpression and inhibition.Mi R-451 a was detected by q RT-PCR after GATA-1 overexpression and inhibition.Results:Under normoxia and hypoxia,the expression of ?-globin and Benzidine staining positive rate and the expression of CD235 a at different time were significantly higher than 0 h(P < 0.01).At 72 h,the expression of ?-globin and benzidine staining positive rate and the expression of CD235 a in hypoxia group were significantly higher than normoxia group(P < 0.05).The expression of GATA-1 and miR-451 a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells(P < 0.05),and were significantly higher than that of the normoxia group at 72 h(P < 0.05).Correlation analysis showed that the expression of GATA-1 was positively correlated with the expression of miR-451 a under hypoxia(P < 0.01).After overexpression of GATA-1 under hypoxia,?-globin,benzidine staining positive cells,and CD235 a and nuclear migration and narrow nuclear cell count at 72 h were significantly higher than negative control group(P < 0.05).After inhibition of GATA-1 under hypoxia,?-globin,benzidine staining positive cells,and CD235 a and nuclear migration and narrow nuclear cell count at 72 h were significantly lower than negative control group(P < 0.05).Compared with the negative control group under hypoxia,the expression of miR-451 a was increased significantly after GATA-1 overexpression(P < 0.05),and the expression of miR-451 a was decreased significantly after the GATA-1 inhibition(P < 0.05).Conclusion:Hypoxia induced the expression of GATA-1 and then up-regulated the expression of miR-451 a that promoted erythroid differentiation of K562 cells.Part ? Expression Relevance of GATA-1 and miR-451 a in Nuclear Red Cells of Patients with HAPC DiseaseObjective: Although chronic hypoxia is the main cause of HAPC,its related molecular mechanism is not clear.This study preliminarily investigated the expression of GATA-1 and miR-451 a in nuclear red cells of HAPC patients,and then conducted correlation analysis to provide experimental data for the mechanism of HAPC.Methods: Peripheral blood of 10 patients diagnosed with HAPC and 10 healthy men were extracted as HAPC group and Control group,respectively.CD34~+ cells,CD71~+ cells and CD235a~+ cells in peripheral blood of HAPC group and Control group were isolated by immunomagnetic beads method.(1)q RT-PCR detected the expression changes of GATA-1 and miR-451 a in CD71~+,CD235a~+ cells.(2)CD34~+ cells isolated from the HAPC group and the Control group and then were cultured under normoxia and hypoxia(1% O2),respectively.Serum-free culture system was used to induce CD34~+ cells to the erythrocyte line.Erythroid Expansion Supplement was added to the induction system to induce 7 D,11 D and 15 D,respectively.q RT-PCR detected ?-globin expression.Meanwhile,the morphological changes of cells were analyzed using Wright-Giemsa staining method,to verify whether erythroid differentiation of CD34 ~+ cells model establishment success.(3)Semisolid clone culture of CD34~+ cell was used to detect cell proliferation.(4)Expression of CD235 a in CD34~+ cells was detected by flow cytometry after 15 D induction.(5)The RNA expression levels of GATA-1 and miR-451 a were detected by q RT-PCR and then the correlation was analyzed.Results:(1)GATA-1 m RNA expression in CD71~+,CD235a~+ cells of HAPC was significantly lower than that in control group(P < 0.01),and miR-451 a expression in HAPC group was significantly higher than that in control group(P < 0.01).(2)In Control group,the expression of ?-globin in normoxia and hypoxia were increased(P < 0.01),and the expression of ?-globin at various time points under hypoxia were significantly higher than that under normoxia(P < 0.01).In HAPC group,the expression of ?-globin under hypoxia was gradually increased(P < 0.01),and the expression of ?-globin at various time points under hypoxia were significantly higher than that under normoxia(P < 0.01);In Control group under normoxic and hypoxic conditions,the number of cells with nuclear migration,nuclear shrinkage and decreased nucleo-cytoplasmic ratio showed an increasing trend(P < 0.05).In HAPC group under normoxic and hypoxic conditions,the number of cells with nuclear migration,nuclear shrinkage and decreased nucleo-cytoplasmic ratio were increased(P < 0.05).These results indicate that the method is feasible to induce CD34~+ cells to differentiate into erythrocytes.(3)On the first day,BFU-E and CFU-E colonies were found in HAPC group under normoxia and hypoxia,but BFU-E and CFU-E colonies were not found in Control group.The number of BFU-E colonies was no significant difference between HAPC group and Control group at 7 D,11 D and 15 D(P > 0.05).The number of CFU-E colonies in the HAPC group at each time point was significantly higher than that in the Control group(P < 0.05).(4)The expression of CD235 a in HAPC group was significantly higher than that in the control group under normoxia and hypoxia(P < 0.05).(5)Under normoxia,the expression of GATA-1 m RNA in HAPC group at 15 D was higher then the Control group(P < 0.01).Under hypoxia,the expression of GATA-1 m RNA in HAPC group at 11 D and 15 D was significantly lower than that in Control group(P < 0.05).Under normoxia,the expression of miR-451 a at 7 D and 15 D were significantly higher than that in the Control group(P < 0.01).Under hypoxia,the expression of miR-451 a in the HAPC group at 11 D and 15 D were significantly higher than that in the Control group(P < 0.01).Correlation analysis showed that GATA-1 in the HAPC group was negative correlated with miR-451 a under hypoxia,r=-0.704(P=0.001).Conclusion: Polycythemia of HAPC may be achieved by increasing the number of CFU-E.The erythroid differentiation of HAPC patients was accelerated.During the differentiation of CD34~+ hematopoietic stem progenitor cells in peripheral blood of HAPC patients to erythroid system under hypoxia,the expression of GATA-1 and miR-451 a were abnormal,and its regulatory relationship may be one of the pathogenesis of plateau erythrocytosis... |
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