| With the development of detection methods,DNA methylation as an epigenetic mechanism has been more and more used in biological research.The occurrence of tumor is associated with the methylation of CpG island.Chronic myeloid leukemia(CML)is a malignant clonal disease originated from hematopoietic stem cells.CML passes through three clinic pathological phases:a chronic phase(CP)、an accelerated phase(AP)and a blastic phase(BP).Patients in chronic stage without treatment will eventually progress to advanced phases(AP and BP)within 3-5 years.A large number of studies have shown that a lot of genetic changes occur during the transformation from CP to AP or BP.SHP-1,as a receptor tyrosine phosphatase,can also act as a key negative regulator in cellular signalling transduction.It was found that the expression of SHP-l gene was decreased or disappeared in various leukemia,malignant lymphoma cell lines and some neoplastic diseases.The reason may be due to the hypermethylation of the promoter region of the SHP-1 gene.It is speculated that methylation of SHP-1 gene plays an important role in the pathogenesis of hematological malignancies.Both the DNA methyltransferase-DNMT1 and the PcG protein complex member-EZH2(Enhancer of zeste homologue 2)take part in a variety of gene methylation processes.DNA methylation is an important epigenetic modification.DNA methyltransferases play a necessary role in gene methylation.EZH2 is an important member of the PcG gene family.EZH2 has been identified as another key epigenetic regulator involved in transcriptional repression in haematological diseases.EZH2 catalyses trimethylation at lysine 27 of histone H3(H3K27me3),which serves as an anchorage point for the recruitment of additional PcG proteins and contributes to the formation of a repressive chromatin state.Thereby,EZH2 can indirectly inhibit the expression of the target gene.Most of these target genes inhibit tumour formation and regulate stem cell differentiation;therefore,silencing these genes would lead to tumour formation.Currently,imatinib is a first-line drug for the treatment of CML.However,some patients are resistant to second-generation kinase inhibitors.Currently,there is limited research on the relationships of DNMT1 and EZH2 activity and SHP-1 methylation in CML.This study explored the relationship between these two factors and SHP-1 methylation to further explore the roles of DNMT1 and EZH2 in the process of blast crisis in CML.This study includes three parts: Part one Expression of SHP-1,DNMT1 and EZH2 gene in chronic myeloid leukemia.Objective:To investigate the relationship between DNMT1,EZH2 and methylation of SHP-1 by detecting the expression of DNMT1,EZH2,SHP-1 gene in CML patients and K562 cell lines and the methylation status of SHP-1 gene in different stages of CML.Methods:Between March 2014 and June 2015,bone marrow(BM)aspirates or peripheral blood(PB)cells were collected from 60 consecutive patients with CML treated at Affiliated Hospital of Hebei University and 10 healthy donors.The patients were divided into CML-CP group,33 cases;CML-AP group,10 cases;CML-BP group,17 cases.The expression of SHP-1、DNMT1、EZH2 in CML BM or PB cells were detected by SYBR Green-based QT-PCR and Western Blot,the levels of SHP-l promoter CpG island methylation status were assessed using methylationspecific polymerase chain reaction(MSP).Statistical analysis was performed with SAS 9.1.3(American SAS Institute Inc.company)software.P<0.05 was statistically significant.Results:1 The levels of SHP-1 mRNASYBR Greenbased QT-PCR analysis showed that the relative levels of SHP-1 mRNA transcripts were 1.49 ± 0.71、1.36 ± 0.65、0.41 ± 0.28、0.28± 0.12 in samples from normal control donors(NC),CML-CP,CMLAP,CML-BP.The levels of SHP-1 mRNA were higher in samples from NC and CML-CP patients than those from CML-AP and CML-BP,P<0.05.There was no significant difference in SHP-1 mRNA expression between NC subjects and CML-CP patients(P>0.05)or between AP-CML and CML-BP patients(P>0.05).2 Expression of SHP-1 protein in CML patientsWestern blot method was used to detect the level of SHP-1 protein in bone marrow mononuclear cells from 10 cases each in 3 different stages of CML.The results showed that the levels of SHP-1protein were higher in CML-CP patients compared to CML-AP and CML-BP,P<0.05.There was no significant difference in SHP-1 protein expression between CML-AP and CML-BP(P>0.05)or between CML-CP and NC.(P>0.05).3 The methylation status of SHP-1 in the clinical casesMethylation of SHP-1 was detected in 7/33 patients with CML-CP,positive rate of 21.2%.However,methylation of the SHP-1 gene was detected in all patients with advanced stage CML,including patients with CML-AP and CML-BP.The positive rate for SHP-1 gene methylation in patients with advanced stage CML was significantly higher compared withpatients with CML-CP(P<0.01).Specifically,4 patients with CML-AP(40%)and 8 patients with CML-BP(47%)exhibited complete SHP-1 promoter methylation,and this difference was not statistically significant(P>0.05).4 The levels of DNMT1 mRNASYBR Greenbased QT-PCR analysis showed that the relative levels of DNMT1 mRNA transcripts were 1.31±0.18、1.75±0.53、2.76±0.78、3.05± 1.23 in samples from normal control donors(NC),CML-CP,CML-AP,CML-BP.The levels of DNMT1 mRNA were higher in samples fromCML-AP and CML-BPpatients than those from NC and CML-CP,P<0.05.There was no significant difference in DNMT1 mRNA expression between NC subjects and CML-CP patients(P>0.05)or between AP-CML andCML-BP patients(P>0.05).5 The levels of EZH2 mRNASYBR Greenbased QT-PCR analysis showed that the relative levels of EZH2 mRNA transcripts were 1.12±0.08、1.25±0.33、1.99±0.78、2.15±1.01、in samples from normal control donors(NC),CML-CP,CML-AP,CML-BP.The levels of EZH2 mRNA were higher in samples fromCML-AP and CML-BPpatients than those from NC and CML-CP,P<0.05.There was no significant difference in EZH2 mRNA expression between NC subjects and CML-CP patients(P>0.05)or between AP-CML andCML-BP patients(P>0.05).6 Correlation of DNMT1,EZH2 and SHP-1 gene expression in CML patientsThe expression of SHP-1 in patients with CML-CP and NC was significantly higher,but decreased gradually in patients with CML-AP and CML-BP.The expression tendency ofDNMT1 and EZH2 is contrary to SHP-1.The expression of DNMT1 and EZH2 in patients with NC and CML-CP was lower,and the expression was significantly higher in the patients with CML-AP and CML-BP.The expression of DNMT1 was negatively correlated with the expression of SHP-1,P<0.05.Similarly,the expression of EZH2 was negatively correlated with the expression of SHP-1,P<0.05.Part two Effects of DNMT1 and EZH2 inhibitors on apoptosis of K562 cell lineObjective:To investigate the effects of DNMT and EZH2 inhibitors on apoptosis of K562 cell line and the associations of DNMT and EZH2 activities with the methylation status of the SHP-1 gene by means of cell culture and flow cytometry.To further explore the significance of DNMT and EZH2 inhibitors in the treatment of CML.Methods:K562 cell lines were cultured and treated with DAC,IM and DZNep,respectively.The effects of drugs on inhibition、apoptosis and cell cycle of K562 cell line were detected by CCK-8 and flow cytometry;The changes of EZH2 expression and SHP-1 methylation status in K562 cell line by DZNep were detected by QT-PCR method,Western blot,methylation specific PCR method.Results:1 CCK8 assay was used to detect the inhibitory rate and median inhibitory concentration(IC50)of K562 cells.The inhibitory rate of K562 cells with DAC and IM increased with the increase of drug concentration and cell culture time.The IC50 value of IM on K562 cell line was 0.25±0.02umol/l(48 hours).The effect of combined application of IM and DAC on inhibition of cell line was higher than that of single application.In addition,the inhibitory rate was 51.06±1.23% after IM combined with DAC(IM 0.25umol/l+DAC100umol/l)for a period of 24 hours,compared with IM alone,half of the time of inhibition was significantly shortened(48 hours).The concentration of 0.1umol/l of IM combined with DAC could make the cell inhibitory rate reach 50.21±1.35% for a period of 48 hours,which was significantly lower than that of IM alone(0.25±0.02umol/l).It can be seen that DAC can promote inhibition of K562 cell line;the combination of DAC and IM has synergistic effect on inhibition of K562 cell line and can reduce the IC50 value of IM.These studies have established a good experimental basis for DAC treatment of CML,especially in advanced stage CML.2 Effect of DAC on apoptosis of K562 cell line by flow cytometryK562 cell lines were cultured with DAC(concentration of 50 umol /l,umol /l,200umol/l)for 48 hours,then the apoptosis was measured.K562 cell line was used as control group.When the concentration of DAC was 50umol/l,the apoptosis rate was 33.62±1.78%;the apoptosis rate was 35.71±1.96% when the concentration was 100umol/l;and the apoptosis rate was 46.99±2.02% when the concentration of DAC was 200umol/l.The apoptosis rate of control group was 30.70±1.58%.So with the increase of DAC concentration,the apoptosis rate increased.There was significant difference between the control group and the treatment group,P<0.05.3 The effect of the two drug combination was greater than that of the single drugK562 cell lines were cultured with DAC(100umol/l),IM(0.25umol/l),IM(0.25umol/l)+DAC(100umol/l)for 24 hours,and the apoptosis of K562 cell line was detected by flow cytometry.The apoptosis rate of K562 cell line was 24.52±1.36%,the apoptosis rate of DAC group was 30.76±1.61%,the IM group was 33.45±1.88%.The apoptosis rate of the combination group was 47.01±2.12%,which was significantly higher than that of the control group and the single drug group.The difference was statistically significant,P<0.05.4 Effect of IM on cell cycleK562 cell lines were added with IM(concentration of 0.05 uM,0.1 uM,0.25uM),we measured the cell cycle after 48 hours.0.05 u M IM group G1 phase accounted for 40.37±1.68%,G2 6.15±1.01%,S 53.48±2.73%;0.1uM IM group G1 48.89±2.74%,G2 4.74±0.61%,S 46.38±1.53%;0.25 uM IM group G1 phase accounted for 80.88±3.04%,G2 0.17±0.05%,S 18.41±1.73%;control group G1 phase accounted for34.76±2.67%,G2 7.51±1.65%,S 57.72±3.68%.So under the action of IM,the ratio of G1 phase increased,the proportion of G2 and S decreased.The change was positively correlated with the dose.5 The effect of DAC on cell cycle was similar to that of IM.The trend was positively correlated with dose.K562 cell lines were added with DAC(100uM,200 u M),and cell cycle was measured after 48 hours.100 uM DAC group G1 phase accounted for 42.81±3.07%,G2 5.83±0.77%,S 51.37±2.51%,200 uM DAC group G1 phase accounted for 62.90±2.96%,G2 1.22±0.27%,S 35.88±2.36%,the control group G1 phase accounted for34.76±2.67%,G2 7.51±1.65%,S57.72 ±3.68%.Under the action of DAC,G1 phase increased,G2 and S decreased.And the change trend was positively correlated with the dose.Similar to IM.6 Effects of DZNep on apoptosis of K562 cell lineThe K562 cells were treated with DZNep(concentration of 10uM).The expression of EZH2 was detected by QT-PCR method and Western blot method for 0 hours,48 hours and 72 hours.In addition,methylation specific PCR method was used to detect the methylation status of SHP-1 gene in the K562 cell line of the dosing group and the non drug group.The results of QT-PCR showed that the relative expression of EZH2 in the K562 group was 2.50± 1.18.When treated with DZNep for 48 hours,the relative expression level of EZH2 decreased to 2.07 ± 1.09,and when cultured for 72 hours,EZH2 dropped to 1.70 ± 0.68.There is a clear downward trend.In addition,the expression of EZH2 protein was significantly decreased after dosing,and the expression of EZH2 decreased with the prolongation of culture time.The SHP-1 gene of K562 cells had a high methylation status,and the methylation status of SHP-1 was decreased after the addition of DZNep.Part three To investigate the relationship between EZH2 and DNMT1 and the promoter region of SHP-1 gene in the progression of chronic myeloid leukemia(CML)Objective:Through cell culture,chromatin immunoprecipitation(ChIP),QT-PCR method,Western blot,methylation specific PCR method,further study on the relationship between DNMT1,EZH2 and SHP-1 gene promoter,and to discuss the role of DNMT1 and EZH2 in the methylation of SHP-1 gene,so as to explore new ideas for the treatment of patients of the advanced stage of CML.Methods: K562 cells were treated with DAC and DZNep respectively,and the expression of DNMT1,EZH2,SHP-1 and the methylation status of SHP-1 gene were detected before and after dosing;ChIP method was used to detect the linkage intensity of SHP-1 promoter region and DNMT1 in K562 cell line before and after adding DAC.The relationship between the methylation of SHP-1 gene promoter region and EZH2 in K562 cell line before and after adding DZNep was detected by ChIP too.Results:1.DNMT1 and SHP-1 expression prior and subsequent to treatment with DACQT-PCR analysis illustrated that the relative mRNA levels of DNMT1 and SHP-1 in K562 cells were 3.54±1.23 and 0.18±0.08,respectively.Treatment with DAC led to a decrease in the DNMT1 mRNA level to 1.55±0.61(P<0.05),but significantly increased the SHP-1 expression level to 1.08±0.20(P<0.05).A similar pattern of the levels of SHP-1 and DNMT1 expression was observed at the protein level.In addition,subsequent to the application of DAC,the methylation level of the SHP-1 gene was decreased in the K562 cells,from complete methylation to partial methylation.2 EZH2 and SHP-1 expression prior and subsequent to treatment with DZNep.QT-PCR analysis demonstrated that the relative mRNA levels of EZH2 and SHP-1 in K562 cells were 2.54±1.20 and 0.18±0.08,respectively.Treatment with DZNep led to a decrease in the EZH2 mRNA level to 1.80±0.62(P<0.05)but a significant increase in the relative mRNA level of SHP-1 to 0.75±0.18(P<0.05).A similar pattern of SHP-1 and EZH2 expression was observed at the protein level.Furthermore,treatment with DZNep led to the progressive demethylation of SHP-1.3 DNMT1 bound to the SHP-1 gene promoter region of K562 cells by ChIPTo demonstrate that DNMT1 is functionally involved in the methylation of the SHP-1 gene promoter and the inhibition of SHP-1 gene expression,ChIP assays using anti-DNMT1 antibodies were performed,and DNMT1 was revealed to be enriched in the SHP-1 promoter region of the K562 cells.Next,drug intervention experiments on K562 cells using the DNA methyltransferase inhibitor DAC were carried out.The expression of SHP-1 increased in the K562 cell line subsequent to DAC treatment.Ch IP detection showed that the DNMT1 expression in the SHP-1 promoter region was decreased in the K562 cell lines treated with drugs.The methylation of the SHP-1 promoter region also decreased subsequent to DAC treatment.4 EZH2 bound to the SHP-1 gene promoter region in K562 cells by ChIP.To identify the epigenetic control of SHP-1 in K562 cells,the role of EZH2 in the regulation of SHP-1 expression was investigated.To evaluate DNA methylation,ChIP assays were performed.In K562 cells,the SHP-1 core region displayed enriched binding to EZH2.To confirm that EZH2 regulates SHP-1 expression,K562 cells were treated with DZNep.Treatment with DZNepwas revealed to increase SHP-1 expression,and the results of the ChIP assay demonstrated reduced binding efficiency of EZH2 to the SHP-1 promoter in the DZNep-treated cells.In addition,DZNep treatment led to decreased SHP-1 methylation.Conclusions:1 The expression levels of SHP-1in CML-AP and CML-BP patients were significantly lower than in CML-CP patients.The expression levels of DNMT1 and EZH2 in CML-AP and CML-BP patients were significantly higher than in CML-CP patients.The expression trend of DNMT1 and EZH2 in each phase of CML was opposite to that of SHP-1.2 MSP detection showed that there was no methylation of SHP-1promoter in the NC group,but the methylation rate of SHP-1 promoter in CML-AP and CML-BP patients was significantly higher than that of CML-CP.It was suggested that the methylation of SHP-1 promoter might decrease the expression level of SHP-1 gene.DNMT1 and EZH2 may be involved in the methylation of SHP-1 gene,which is closely related to the progression of chronic myeloid leukemia.3 DAC could promote the apoptosis of K562 cell line.The combination of DAC and IM had synergistic effect on apoptosis,and which could reduce the IC50 value of IM.The proportion of G1 phase in K562 cellsincreased with the effection of DAC and IM,the proportion of G2 and S decreased,and which was positively correlated with the dose.The combination of the two drugs was more effective than single drug on apoptosis of K562 cell lines.4 The expression of EZH2 at the molecular and protein level decreased after adding DZNep to K562 cell line,and there was significant difference with NC.The SHP-1 gene of K562 cells had a high methylation status,and the methylation status of SHP-1 gene was decreased after the addition of DZNep.After K562 cell lineswere cultured with DNMT1 inhibitor,the expression of SHP-1 protein and gene level was increased,the expression of DNMT1 gene and protein were decreased,and the degreemethylation of SHP-1 was decreased.After K562 cell lineswere cultured with DZNep,the expression of SHP-1 protein and gene level was increased,the expression of EZH2 gene and protein were decreased,and the degreemethylation of SHP-1 was decreased.5 Both DNMT1 and EZH2 were associated with the promoter region of SHP-1 gene,which was involved in the methylation of SHP-1 gene and associated with the progression of CML disease. |
PDF Full Text Request