Research On The Epigenetic Regulation Mechanism Of The PTPN6 Gene In Advanced Chronic Myeloid Leukemia

Chronic myelogenous leukemia(CML)is a malignant disease of hematopoietic stem cell.It is the most common chronic leukemia in China,accounting for 15% to 20% of adult leukemia.The natural course is divided into the chronic phase(CP),the accelerated phase(AP)and the blast crisis(BP).The accelerated phase and the blast phase are considered to be the progression of the disease.t(9;22)(q34;q11)translocation forms Philadelphia chromosome and its lead to the formation of BCR/ABL fusion genes,which encodes P210BCR/ABL protein with strong tyrosine kinase activity.It leads to the continuous phosphorylation of a series of signal proteins,affecting the proliferation,differentiation and apoptosis of cells,and is the molecular basis of the pathogenesis of CML.More than 10 years ago,tyrosine kinase inhibitors(TKIs)have greatly improved the prognosis of patients with CML.However,with the expansion of clinical application and the passage of time,some patients have drug resistance and disease progression,the treatment effect is poor,and the mortality rate is very high.To overcome this problem,people actively seek new therapeutic targets and combined use of other drugs.Tumorigenesis is related to activation of proto oncogene and inactivation of tumor suppressor gene.Inactivation of tumor suppressor genes is regulated by genetics and epigenetics.Epigenetics refers to the sequence of DNA does not change the gene expression that heritable changes,and in the process of cell growth and proliferation in stable transmission,including DNA methylation,histone modification,chromatin remodeling and RNA interference.PTPN6(also termed SHP-1),a cytoplasmic protein tyrosine phosphatase family,plays a negative role in cell signal transduction,and is negatively correlated with tumor formation and growth.It has been found that methylation of PTPN6 promoter leads to low expression in CML patients.Methylation and histone modification of tumor suppressor genes are the most common epigenetic regulation of tumor cells and play an important role in tumorigenesis and development.The methylation of gene promoter can directly inhibit the binding of transcription factors to the promoter and inhibit gene transcription.Histone acetyltransferase(HATs)and histone deacetylase(HDACs)regulate histone acetylation.HDACs can inhibit the binding of transcription factors and DNA by reducing the level of histone acetylation,thereby inhibiting the transcription and affecting cell differentiation and apoptosis.The aim of this study is to provide a new way to capture the progress of CML by studying the relationship between PTPN6 gene and CML progression,and the regulation and mechanism of methylation and histone deacetylation on PTPN6.This research paper is divided into three parts: Part one: Study on the mechanism of aberrant methylation of PTPN6 gene in patients with chronic myelogenous leukemiaObjective: Detection of PTPN6 gene promoter methylation,and PTPN6,DNMT1,DNMT3 a,DNMT3b,MeCP2,MBD2 and HDAC1 gene mRNA and protein expression level,to explore the progress of PTPN6 gene and CML and the relationship between the methylation and histone deacetylation of its regulation.Methods: From September 2014 to October 2015,44 cases of bone marrow or peripheral blood samples from patients with chronic myeloid leukemia were selected from the Department of Hematology and Pediatrics at Hebei Medical University.They were divided into three groups according to the stages of the disease.10 cases of peripheral blood samples from healthy volunteers were selected.The methylation status of PTPN6 gene promoter region Cp G island was detected by MSP,and the expression of PTPN6,DNMT1,DNMT3 a,DNMT3b,MeCP2,MBD2 and HDAC1 genes mRNA and protein were detected by PCR and Western Blot.Results:1 Methylation of PTPN6 gene promoter in CML patientsNC group were not detected the DNA promoter methylation of PTPN6 gene.20 cases of CML-CP patients in 5 cases were detected the methylation,the positive rate was 25%.24 cases of CML patients with disease progression,including 14 cases of CML-AP and 10 cases of CML-BP patients,PTPN6 gene were detected the promoter methylation.The positive rate of the methylation of the PTPN6 gene promoter in patients with CML disease progression was significantly higher than the NC group and CML-CP group,with statistical significance(P<0.01).The positive rate of methylation of PTPN6 gene promoter in patients with NC and CML-CP was not statistically significant,P=0.14.2 Expression of mRNA level in CML patientsThe expressions of DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 mRNA in CML patients were higher than that of NC group,and the expression PTPN6 mRNA was lower than that of NC group.There have statistically significant difference in these groups,P<0.05.There were no significant differences in the expression of DNMT3 b mRNA between the CML patients groups and the NC group,P>0.05.Among them,the expression of DNMT3 a,MeCP2,MBD2 and HDAC1 mRNA in the CML-BP group was the highest,the difference was statistically significant,P<0.05.The expression of PTPN6 mRNA was the highest in group CML-CP,the difference was statistically significant.There was no significant difference between CML-AP and CML-BP group(P>0.05).3 Expression of protein level in CML patientsThe expression of DNMT1 protein was low in group NC,and higher in CML group.The difference was statistically significant(P<0.05),and there was no significant difference in the expression between the patients(P>0.05).The expression of DNMT3 a,MeCP2,MBD2 and HDAC1 protein were lower in group NC,and higher in CML group,and highest in group CML-BP,with statistical difference(P<0.05).The PTPN6 protein was highly expressed in group NC,and lower expression in CML group,and lowest expression in group CML-BP,and statistically significant difference(P<0.05).The expression level of DNMT3 b protein were not statistically different between the NC group and the CML group,P>0.05.Conclusion:The low expression of PTPN6 gene in the patients with advanced CML is related to hyper-methylation of promoter region and may be regulated by DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 genes.Part two: the effect and mechanism of demethylation drugs and histone deacetylase inhibitors on PTPN6 gene in K562 cells and KCL22 cellsObjective: Demethylation drugs and histone deacetylase inhibitors treatment of KCL22 cells and K562 cells,detect the expression of PTPN6 gene promoter Cp G islands methylation,and the changes of PTPN6,DNMT1,DNMT3 a,DNMT3b,MeCP2,MBD2 and HDAC1 mRNA and protein.To investigate the methylation and histone modification mechanism of regulation of PTPN6 gene in CML blast cell line.Methods:The KCL22 cells and K562 cells were treated with different concentrations of demethylation drugs(Decitabine and 5-Azacytidine)and histone deacetylase inhibitors [(valproic acid,VPA)and LBH589].Bisulfite sequencing PCR(BSP)was used to detect the methylation alteration of PTPN6 gene promoter CpG Island.PCR and Western Blot were used to detect the alteration of PTPN6,DNMT1,DNMT3 b,MeCP2,DNMT3 a,MBD2 and HDAC1 mRNA and protein expression.Results:1 Detection of PTPN6 gene promoter methylation in the drug treated cells by MSP assayIn K562 cells,methylation of PTPN6 gene promoter CpG was 80.9% before drug treatment.After VPA treatment was 56.4%,and the difference was statistically significant,P<0.05.After LBH589 treatment was 80.9%,5-Azacytidine was 78.2%,Decitabine was 81.8%.The difference between the three groups was not statistically significant(P>0.05).In KCL22 cells,methylation of PTPN6 gene promoter Cp G was 85.5% before drug treatment.After VPA treatment was 66.4%,and after Decitabine treatment was 72.7%.The difference between the two groups before treatment was statistically significant(P<0.05),but there was no significant difference between the two groups(P>0.05).LBH589 treatment was 82.7%,5-Azacytidine was 77.3%,and the difference between the two groups was not statistically significant(P>0.05).2 Changes of mRNA expression after drug treatment with the cells1)VPA treatment of K562 cellsIn K562 cells,after treatment,the expression of DNMT1,MeCP2 and MBD2 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.The DNMT3 a and HDAC1 mRNA decreased obviously,the difference between the low concentration group and the middle and high concentration group was statistically significant,P<0.05,and there was no statistical difference between the middle concentration and the high concentration group(P>0.05).There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).In KCL22 cells,after treatment,expression of DNMT1,DNMT3 a and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.The expression of MBD2 mRNA decreased,the difference was statistically significant(P<0.05),but there was no significant difference between the groups(P>0.05).There was no significant difference in the expression of DNMT3 b and MeCP2 mRNA with the treatment(P>0.05).2)LBH589 treatment of K562 cellsIn K562 cells,after treatment,the expression of DNMT3 a,MeCP2 and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.The expression of DNMT1 and MBD2 mRNA decreased significantly,the difference was statistically significant(P<0.05),but there was no significant difference between the different concentration groups(P>0.05).There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).In KCL22 cells,after treatment,the expression of DNMT1 and MBD2 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.The expression of HDAC1 mRNA decreased(P<0.05),but there was no significant difference between the groups(P>0.05).The expressions of DNMT3 a,DNMT3b and MeCP2 mRNA were not significantly different with the treatment(P>0.05).3)5-Azacytidine treatment of K562 cellsIn K562 cells,after treatment,the expression of MeCP2,MBD2 and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.The expression of DNMT1 and DNMT3 a mRNA decreased significantly,the difference was statistically significant(P<0.05),but there was no significant difference between the different concentration groups(P>0.05).There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).In KCL22 cells,after treatment,the expression of DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).4)Decitabine treatment of K562 cellsIn K562 cells,after treatment,the expression of DNMT3 a,MeCP2,MBD2 and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly.The highest concentration group showed the most significant difference,the difference was statistically significant,P<0.05.The expression of DNMT1 mRNA decreased significantly,the difference was statistically significant(P<0.05),but there was no significant difference between the groups(P>0.05).There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).In KCL22 cells,after treatment,the expression of DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 mRNA decreased significantly,and the expression of PTPN6 mRNA increased significantly,the highest concentration group was statistically significant,the difference was statistically significant,P<0.05.There was no significant difference in the expression of DNMT3 b mRNA with the treatment(P>0.05).3 Protein levels of the CML blast cell line after drug treatmentIn K562 and KCL22 cells,after treatment with the drugs,the expression of DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 protein were significantly decreased,the difference was statistically significant(P<0.05),PTPN6 protein expression was significantly increased,the difference was statistically significant(P<0.05),DNMT3 b protein expression was no significant difference(P>0.05).In K562 cells,the expression of DNMT3 a and MBD2 protein decreased significantly after treatment with VPA and 5-Azacytidine,and the difference was statistically significant(P<0.05),but there was no significant difference between the two drugs(P>0.05).After treatment with 5-Azacytidine,the decrease of MeCP2 protein was the most obvious,and the difference was statistically significant(P<0.05).After treatment with VPA,the amount of PTPN6 protein increased at least,and the other three groups increased significantly,the difference was statistically significant(P<0.05),but the difference between the three groups was not statistically significant(P>0.05).In KCL22 cells,the expression of MeCP2 and MBD2 protein decreased significantly after treatment with 5-Azacytidine and Decitabine,and the difference was statistically significant(P<0.05),but there was no significant difference between the two groups(P>0.05).After 5-Azacytidine treatment,the expression of DNMT3 a and HDAC1 protein decreased significantly,the difference was statistically significant(P<0.05).After LBH589 and Decitabine treatment,the expression of PTPN6 protein increased significantly,the difference was statistically significant(P<0.05),but the difference between the two groups was not statistically significant(P>0.05).Conclusion:1 In CML blast cells KCL22 and K562,the promoter methylation of PTPN6 was high,and the expression was low.It may be related to DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 genes.2 After the demethylation drugs and histone deacetylase inhibitors were used,the methylation level of PTPN6 gene decreased,the expression level increased,and the expression levels of DNMT1,DNMT3 a,MeCP2,MBD2 and HDAC1 decreased.3 Demethylation drugs have the effect of histone deacetylase inhibitors,and histone deacetylase inhibitors also have demethylation effects.5-Azacytidine showed strong histone deacetylase inhibitor effect,and VPA showed stronger demethylation effect.Part three: the study on the regulation of PTPN6 gene by HDAC1Objective: To investigate the regulatory mechanism of HDAC1 on PTPN6 gene.Methods: In KCL22 cells,the mouse IgG antibody was used as a control antibody,and CoIP-MS was used to detect proteins bound to PTPN6 proteins and analyzed by mass spectrometry.ChIP-seq detected the gene associated with HDAC1 protein and sequenced analysis.Results:1 Results of the CoIP-MS561 identified proteins were pulled down by the IP antibody in the experimental group,393 identified proteins were pulled down in the control antibody group.300 proteins were detected separately in the experimental group,that is to say,300 proteins interacting with the PTPN6 protein.The 300 proteins were identified by mass spectrometry,and the direct binding of HDAC1 and the PTPN6 protein was determined.Results showed that the PTPN6 protein is mainly involved in cell survival and death,protein synthesis,cell growth and proliferation,cell function and maintenance of biological processes,and is closely related with the EIF2 signalling pathway,the protein ubiquitination pathway and the cell cycle signalling pathway,CD437,RICTOR,MYC and other upstream factors associated with tumour and tissue injury and genetic diseases,and malignant haematological diseases were closely related.2 Results of the ChIP-seqAfter the samples were sequenced by Ch IP-seq,the average output of the original reads was 27588525.By controlling Clean Reads for 26510483 and scanning Peak(ChIP Sequencing enrichment regions)in the genome wide range,2985 genes,including MAPK,AKT and STAT5 genes,were obtained.Analysis of gene function components showed that introns accounted for 36.7%,exons 4.1%,upstream regulatory elements 4.9%,and downstream regulatory components 2%.The pathway analysis of the enriched gene showed that it was closely related to the MAPK/ERK,PI3K/Akt/NF-,NF,B,JAK/STAT and c-myc signaling pathways.Conclusion:In the CML blast cell line KCL22,MAPK,AKT,STAT5 gene may have regulatory effect on the expression of HDAC1 protein,HDAC1 protein may exist regulation effect on the expression of PTPN6 protein,the specific mechanism is to further research.In conclusion,the PTPN6 gene is closely related to the progression of CML.In the progressive phase of CML,hyper-methylation of the PTPN6 gene promoter region results in suppression of its expression.Methylation and histone deacetylation are involved in the regulation of aberrant methylation of PTPN6.The possible mechanism is that the MAPK,AKT,and STAT5 genes promote the formation of HDAC1 transcription complexes and play a bridge role between MeCP2 and MBD2,thus enabling DNMT1 and DNMT3 a to play a role in methylation of PTPN6 genes.Histone deacetylase inhibitors are expected to be used in the treatment of CML progression.