The Role Of PTPN21 In Cell Proliferation And Drug Resistant Of Acute Lymphoblastic Leukemia

Background:Acute Lymphoblastic Leukemia(ALL)is a malignancy characterized with abnormal proliferation of immature lymphocytes in bone marrow,peripheral blood and other organs.Poor long-term prognosis and low survival rate after relapse are main problems in adult ALL patients.Genetic alterations play pivotal roles in the carcinogenesis of ALL.The exploration of genetic alterations contributes to not only the definition of pathogenesis in ALL,but also the direction of clinical therapy.Increasing evidence has revealed that mutation and abnormal expression of PTKs and PTPs induced the dysfunction of phosphorylation and dephosphorylation,which took part in pathogenesis of ALL via promotion of proliferation,abnormal differentiation and apoptosis resistance.PTPN21 encodes a protein named protein tyrosine phosphatase,non-receptor type 21.Previous reports have uncovered the high expression of PTPN21 in tumor tissues,which was relevant to the growth of tumor cells.However,whether PTPN21 is overexpressed in ALL is not clear yet.The role of PTPN21 in the pathogenesis of ALL is required to be definited.This study discovered the high expression of PTPN21 in ALL samples.We aimed to explore the effects of alteration of PTPN21 expression on cell apoptosis,cell proliferation and drug resistance and relevant mechanisms through overexpression by lentiviral infection and knockout by CRISPR-Cas9.Part 1 Role of PTPN21 overexpression in cell proliferation and drug resistance of acute lymphoblastic leukemiaObjective:To clarify the role of PTPN21 in pathogenesis of acute lymphoblastic leukemia by exploring whether overexpression of PTPN21 has effects on cell proliferation and drug resistance.Methods:The mRNA expression of PTPN21 in bone marrow samples of ALL patients and control group was assessed by real time PCR.The overexpression of PTPN21 in Jurkat,NALM-6 and Reh cells was acquired by lentiviral infection.The validation of PTPN21 overexpression was detected by Western blotting and immunofluorescence.The flow cytometry analysis was used to detect apoptosis and cell cycle.The phosphorylation of Src and MAPK signal pathways was assessed by Western blotting.Small-molecule inhibitors of MAPK pathways were applied to definite the role of relevant signal pathways.Drug resistance was analyzed depending on the apoptosis of cells pretreated with daunorubicin detected by flow cytometry.Digital Gene Expression Tag Profiling(DGE)sequencing was used for the screening of differentially expressed genes.The expression of screened genes was confirmed by real time PCR.Results:The mRNA expression of PTPN21 in ALL patients was abnormally high,especially in newly diagnosed and relapse patients.However,there was no significant difference between complete remission patients and control group.In Jurkat,NALM-6 and Reh cells,the cell ratio of GO phase of overexpression group was significantly lower than that of control group.The cell ratio of S/G2/M phases of overexpression group was significantly higher than that of control group.There was no significant difference between cell ratios of G1 phase of these two groups.PTPN21 overexpression promoted the proliferation of Jurkat,NALM-6 and Reh cells.The phosphorylation of Src in PTPN21-overexpressed Jurkat cells distinctly decreased and the phosphorylation of ERK1/2 and JNK distinctly increased.There was no obvious change in the phosphorylation of p38 MAPK.As to NALM-6 cells,the phosphorylation of Src in overexpession group distinctly decreased and the phosphorylation of ERK1/2 and p38 MAPK distinctly increased.No obvious change was observed in the phosphorylation of JNK.In Reh cells,the phosphorylation of Src in overexpession group distinctly decreased and the phosphorylation of ERK1/2,p38 MAPK and JNK distinctly increased.PD98059,the inhibitor of ERK1/2 pathway,had no effects on the promotion of cell proliferation regulated by PTPN21 in ALL cells.SP600125,the inhibitor of JNK,could inhibit the PTPN21-induced promotion of Jurkat cells effectively.However,SP600125 had no effects on the promotion of cell proliferation in Reh cells.SB203580,the inhibitor of p38 MAPK,could inhibit the PTPN21-induced promotion of NALM-6 cells effectively and could partly inhibit that of Reh cells.PTPN21 suppressed the apoptosis of Jurkat and NALM-6 cells induced by daunorubicin.The elevated expression of IL22RA1 was detected in cells overexpressed PTPN21.Conclusion:The high expression of PTPN21 may play roles in leukemogenesis and relapse of ALL.PTPN21 activated Src by dephosphorylation and subsequently activated downstream MAPK signal pathways,which finally promoted the proliferation of ALL cells.The involved MAPK signal pathways varied in different cell lines.JNK pathway and p38 MAPK pathway were pivotal mechanisms of Jurkat and NALM-6 cells,respectively.P38 MAPK pathway may partly regulate the promotion of cell proliferation by PTPN21.PTPN21 inhibited daunorubicin-induced apoptosis of ALL cells,which reflected drug resistance.The elevated expression of IL22RA1 may play roles in the drug resistance regulated by PTPN21.Part 2 Effects of PTPN21 knockout in Jurkat cells by CRISPR-Cas9 on cell proliferationObjective:To clarify whether knockout of PTPN21 by CRISPR-Cas9 system inhibits cell proliferation of Jurkat.Methods:CRISPR Design tool of Zhanglab MIT was applied to design gRNA of CRISPR-Cas9 system targeting exon 2 or exon 13 of PTPN21.The plasmid pX458 was used as expression vector of gRNA.The cutting efficiency of different gRNAs was assessed by T7E1 assay in 293T cells.Nucleofection was applied to transfect Cas9 plasmids into Jurkat cells.GFP positive cells were selected by flow cytometry.Extreme dilution was used to acquire single-cell clones of transfected Jurkat cells.Genomic DNA was extracted and amplified by PCR.The PCR products were sequenced.TA clone was used to clarify the specific DNA sequences of clones with double signals.The apoptosis and cell cycle of single-strand and double-strand knockout clones were detected by flow cytometry.Results:The gRNAs targeting exon 2 and exon 13 were respectively selected with highest cutting efficiency by T7E1 assay.We acquired 5 single-allele knockout clones and 6 double-allele knockout clones.The knockout of PTPN21 had no effects on apoptosis of Jurkat cells.The cell ratio of S/G2/M phases of knockout clones was significantly lower than that of negative control,which implied that knockout of PTPN21 inhibited the proliferation of Jurkat cells.Conclusion:We established PTPN21-knockout cell lines of Jurkat.The proliferation of Jurkat cells was inhibited by knockout of PTPN21.