| Objective:Desitabine(DAC)has made great progress in the treatment of acute myeloid leukemia(AML)in recent years,but due to primary resistance or secondary resistance during treatment with DAC,some patients lead to treatment failure and disease progression.Our previous study found that ID1 may be involved in drug resistance to DAC.In this study,the methods of overexpression and intervention of ID1 expression were adopted to analyze the influence of ID1 on the biological function of leukemia cells and explore its mechanism of action in drug resistance to DAC.Methods:(1)Using gene overexpression technology,ID1 was transfected into leukemia cell lines K562 and HL60 by lentivirus to obtain K562 and HL60 cells(ID1-K562,ID1-HL60)and their control cells(NC-K562,NC-HL60);Using gene silencing technology,the shID1 gene was transfected into DAC-resistant K562 cell line(K562/DAC)by lentivirus.After screening,ID1 silenced K562/DAC cells(sh ID1-K562/DAC)and their control cells(shNC-K562/DAC);Realtime quantitative PCR(RQ-PCR)and Western blot were used to detect the expression level of ID1 in transfected cell lines.(2)Using cell counting method and Annexin V-PE/7-AAD to analyze the expression of ID1 on the growth and apoptosis of transfected cells.(3)We treated ID1-K562,ID1-HL60,shID1-K562/DAC cells and control cells with different concentrations of DAC respectively,then counted the number of viable cells using trypan blue exclusion staining method,and used SPSS to calculate the half inhibitory concentration inhibitory concentration(IC50),to analyze the effect of ID1 expression on DAC resistance in leukemia cells and drug-resistant cells.(4)Inoculate NC-K562 and ID1-K562 cells subcutaneously on the back of nude mice to establish a model of subcutaneous xenograft tumor in nude mice and treat with DAC.At the same time,NS group was set up.Observation indicators include mouse weight,survival time,and drug sensitivity and so on.Nude mice were sacrificed to take subcutaneous tumor tissue,and the changes of ID1 expression were detected by RQ-PCR and Western blot.The tumor tissues were taken for HE staining to detect their pathological conditions.(5)The expression of ID1 in K562 cells treated with different concentrations of DAC was detected by RQ-PCR,and the methylation level of ID1 promoter region was detected by BSP.Establish K562/DAC cells overexpressing miR-29 b and their control cells(NC-K562/DAC)to analyze the expression of ID1;co-culture with DACs of different gradient concentrations,and count the surviving cells using trypan blue staining method.Calculate the IC50 value of each group using SPSS software.ID1 was analyzed as a target gene of miR-29 b,and miR-29b-ID1 signaling pathway was explored to be involved in the resistance of leukemia cells to decitabine.Results:(1)The effect of ID1 overexpression on the biological function of leukemia cellsThe expression of ID1-K562 and ID1-HL60 after lentivirus transfection was detected using RQ-PCR and Western blot.The results showed that the expression of ID1 in the ID1-K562 and ID1-HL60 groups increased(P<0.05).The results of cell growth experiments showed that compared with the control,the cell growth rate of the ID1-K562 and ID1-HL60 groups was significantly increased(P <0.05);the early apoptosis was significantly reduced(P <0.05).K562,NC-K562,ID1-K562 and HL60,NC-HL60,ID1-HL60 were treated with different concentrations of decitabine.The IC50 was calculated.The results showed that compared with K562 and NCK562 groups,the resistance of ID1-K562 group to DAC increased significantly(P<0.05);compared to HL60 and NC-HL60,the resistance of ID1-HL60 group to DAC increased significantly(P<0.05).It shows that ID1 can promote the resistance of K562 and HL60 to DAC.In order to explore the effect of overexpression of ID1 on tumor growth and DAC resistance in nude mice,a nude mouse leukemia cell subcutaneous transplantation tumor model was established.The results showed that compared with the NS group injected with NC-K562,the tumor volume increased in the NS group injected with ID1-K562(P<0.05);suggesting that ID1 may promote the growth of subcutaneous tumors in nude mice.Compared with the NC-K562 injected DAC group,the tumor volume increased in the ID1-K562 injected DAC group(P<0.05),suggesting that ID1 may increase the resistance of leukemia K562 cells to DAC in nude mice.(2)Biological effects of ID1 interference on DAC-resistant leukemia cellsCompared with the control group,the expression level of ID1 in shID1-K562/DAC group was reduced.The results of cell proliferation experiments showed that compared with the control group,the growth rate of cells in the ID1 silencing group was reduced,which was statistically significant(P <0.05).The shNC-K562/DAC and shID1-K562/DAC cells were treated with different concentrations of DAC for three days,the number of surviving cells was counted,and the IC50 value of each group of cells was calculated using SPSS software.The results showed that the IC50 of shNC-K562/DAC and shID1-K562/DAC were(2.25±0.19)μM/L and(1.13±0.13)μM/L,respectively,which was statistically significant(P <0.05).It is suggested that the survival rate of K562/DAC cell lines silenced by ID1 is lower than that of the control group,and ID1 silencing can increase the sensitivity of K562/DAC cells to DAC.(3)Effect of miR-29b-ID1 signal on the biological function of K562 / DACUsing RQ-PCR method to detect the expression of ID1 in K562 cells treated with different concentrations of DAC,the results showed that compared with the K562 cells without DAC treatment,the expression of ID1 in K562 cells treated with DAC was significantly reduced;The expression of ID1 was up-regulated,suggesting that there is abnormal regulation of ID1 expression in DAC-resistant cells.BSP sequencing method was used to detect the methylation level of ID1 promoter region.The results showed that ID1 in K562 cells was completely unmethylated.The promoter region of ID1 in DAC-treated K562 was highly unmethylated which indicated that the expression of ID1 was not regulated by methylation.ID1 may participate in DAC resistance by regulating upstream miR-29 b of ID1.To this end,lentiviral transfection technology was also used to construct miR-29 b overexpression cell lines(miR-29b-K562/DAC)and corresponding control groups(NC-K562/DAC).NC-K562/DAC and miR-29b-K562/DAC cells were treated with different concentrations of DAC for three days,the number of surviving cells was counted,and the IC50 value of each group of cells was calculated using SPSS software.The results showed that the IC50 of the miR-29bK562/DAC group was(0.72±0.03)μM/L,and the control group was(2.17±0.03)μM/L;compared with the control group,the sensitivity of miR-29b-K562/DAC cell group to DAC increased and the drug resistance decreased.The expression levels of ID1 in the two cell lines were detected by RQ-PCR and Western blot,respectively.The results showed that the levels of ID1 mRNA and protein in miR-29b-K562/DAC were reduced.It is suggested that overexpression of miR-29 b may increase the sensitivity of DAC by reducing the expression of ID1 in K562/DAC cell line.Conclusion:(1)ID1 can promote the growth of leukemia cells and reduce early apoptosis;(2)ID1 can reduce the sensitivity of leukemia cells to DAC;(3)The expression of ID1 is not regulated by methylation,the expression of ID1 in miR-29 b overexpressing cells is reduced,and miR-29b-ID1 signaling may be involved in DAC resistance. |
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