Regulation Mechanism Of ?-globin Expression In K562 Cells Induced By Hydroxyurea Through P38MAPK Signaling

Objective: To investigate the regulation and mechanism of hydroxyurea on the expression of ?-globin in K562 cells through P38 MAPK signaling and to provide a new experimental basis for clarifying the mechanism of ?-globin synthesis induced by hydroxyurea.Methods: K562 cells were induced by hydroxyurea with final concentrations of 0?M,100?M,200?M,300?M,and 400?M,and four different time points(24h,48 h,72h,and 96h)were selected for examination,respectively.RT-PCR was used to detect the expression levels of ?-globin gene mRNA and CCK-8 kits were used to detect the K562 cells survival rate at different concentrations hydroxyurea induced different time.Selecting the optimal concentration and time of hydroxyurea to evaluate the differences in expression of relevant transcription factors such as BCL11 A gene after treatment by hydroxyurea.Subsequently,four groups were set up,including hydroxyurea group,hydroxyurea+SB203580 group,equal volume SB203580 inhibitor group and PBS negative control group.To further detect mRNA expression levels of?-globin gene,P38 MAPK gene and relevant transcription factors gene byRT-PCR as well as to test the expression levels of ?-globin,total P38 MAPK protein and phosphorylation P38 MAPK protein by Western blot analysis.Rusults: 1.Selecting optimal regulated concentration and induced time of hydroxyurea.The results showed there was a significant difference in the levels of ?-globin gene mRNA at different concentrations and time(F=352.387,P=0.000;F=823.875,P=0.000).And there was an interaction between concentration and time(F=303.642,P=0.000).The concentration of 300?M for72 h had the strongest effect on ?-globin gene mRNA expression,followed by400?M for 96 h.2.The effect of hydroxyurea on the survival rate of K562 cells was detected by CCK-8 kits.The cells survival rate was affected by both time(F=464.901,P=0.000)and concentration(F=196.683,P=0.000),and there was an interaction between the two factors(F=11.779,P=0.000).With the increase of the hydroxyurea concentration and the prolongation of the action time,the survival rate of K562 cells gradually decreased.There was a significant difference in the cells survival rate between each time point at the same concentration and different concentrations at the same time(P=0.000).3.The effect of hydroxyurea on the apoptosis rate of K562 cells was detected by flow cytometry.The results showed there were significant difference in the early apoptosis rate,late apoptosis rate and total apoptosis rate at different concentrations of hydroxyurea(F=3.802,P=0.039,F=6.298,P=0.043,F=7.709,P=0.004).The apoptosis rate of K562 cells had concentration dependent,and the higher the hydroxyurea concentration,the higher the apoptosis rate.4.The expression levels of relevant transcription factor genes mRNA in K562 cells after treated with hydroxyurea.The expression levels of BCL11 A mRNA was significantly decrease,which was 0.21-fold of control group(P=0.000).However,the mRNA expression levels of GATA1,TAL1,SOX6 and KLF1 were not significantly different from those in the control group(P=0.052,P=0.115,P=0.590,P=0.447).5.The expression levels of ?-globin gene mRNA in K562 cells of hydroxyurea group,hydroxyurea+SB203580 group and other treatment groups.The expression of ?-globin mRNA in hydroxyurea group was significantly higher than that in other groups(P=0.000),which were 5.75-fold,3.48-fold and8.21-fold higher in the control group,hydroxyurea+SB203580 group and SB203580 group,respectively.While there was no significant difference between hydroxyurea+SB203580 group and SB203580 group compared with the control group(P=0.052,P=0.324).6.The expression levels of ?-globin in K562 cells of hydroxyurea group,hydroxyurea+SB203580 group and other treatment groups.The expression of?-globin in the hydroxyurea group was significantly higher than that in other groups,which was 1.42-fold(P=0.004),2.06-fold(P=0.000)and 1.74-fold(P=0.000)that of the control group,the hydroxyurea+SB203580 group and the SB203580 group,respectively.There was no significant difference between SB203580 group and control group and hydroxyurea+SB203580 group(P=0.119,P=0.260).7.The expression levels of P38 MAPK gene mRNA in K562 cells of hydroxyurea group,hydroxyurea+SB203580 group and other treatment groups.RT-PCR results showed that there was no significant difference in the levels of P38 MAPK gene mRNA in each group(P=0.234).8.The expression levels of total P38 MAPK protein and phosphorylated P38 MAPK protein in K562 cells of hydroxyurea group,hydroxyurea+SB203580 group and other treatment groups.Results fromwestern blot showed that there was no difference in the total P38 MAPK protein level was found among the different treatment groups(P=0.132).However,the expression of phosphorylated P38 MAPK protein were significantly different among the different treatment groups(P=0.000).The phosphorylation P38 MAPK protein in hydroxyurea group was significantly higher than that in other groups(P=0.000),and there was no significant difference between the other groups.9.The expression levels of relevant transcription factors gene mRNA in K562 cells of hydroxyurea group,hydroxyurea+SB203580 group and other treatment groups.Among the different groups the levels of BCL11 A mRNA had a significant difference(P=0.000).The expression level of BCL11 A mRNA in the hydroxyurea group was significantly lower than that in the other groups(P=0.000),which were 0.38-fold,0.31-fold and 0.28-fold of the control group,the hydroxyurea+SB203580 group and the SB203580 group,respectively.There was no significant difference between the hydroxyurea+SB203580 and SB203580 groups(P=0.237).While there was no significant difference in the expression levels of KLF1,GATA1,SOX6 and TAL1 gene mRNA in each group(P=0.249,P=0.126,P=0.981,P=0.476).Conclusion:1.The study showed that hydroxyurea could promote the ?-globin gene mRNA expression in K562 cells by inhibiting the expression of BCL11 A gene.2.The study showed that hydroxyurea could activate the P38 MAPK signaling by increasing the phosphorylation level of P38 MAPK to promote the?-globin gene mRNA and ?-globin expression in K562 cells.3.The study confirmed that hydroxyurea could inhibit the expression of BCL11 A gene to promote the ?-globin expression in K562 cells by activatingP38 MAPK signaling.