The Significance And Expression Of SHIP Gene And4EBP1Gene In Chronic Myeloid Leukemia Patients

Objective: Chronic myelogenous leukemia (CML) is a pluripotenthematopoietic stem cell malignant proliferative diseases, Ph chromosomeproduces BCR/ABL fusion gene, its tyrosine kinase activity was significantlyenhanced and activation of oncogenes which affect cell proliferation,differentiation, apoptosis, inhibition of normal hematopoiesis and causedisease. Tyrosine kinase inhibitors (tyrosine kinase inhibitors, TKIs) aretargeted therapy, which against the BCR/ABL. However some patientsdevelop resistance. SHIP gene mainly expressed in the hematopoietic system,which is an important negative regulator of hematopoietic, which play a roleby inhibiting the PI3K/Akt pathway. Significance of this project is to furtherstudy the role of SHIP gene in leukemia pathogenesis and provide atheoretical basis for the search for new therapeutic targets of leukemia.SHIP (SH2-containing inositol polyphosphate5-phosphatase) mainlyexpressed in the hematopoietic system, involved in the regulation of normalhematopoietic cells, particularly myeloid cell, which is an important negativeregulator of the PI3K/Akt pathway. SHIP degraded PIP3intophosphatidylinositol3,4-bisphosphate [PI (3,4) P2] by catalyzingphosphatidylinositol D-5Phosphate dephosphorylation, thus reduced signaltransduction in downstream of PI3K. SHIP can inhibit the PI3K/Akt/GSK-3βpathway which affect cell cycle and cell will arrest at G1phase[1]. Recentstudies have shown that, SHIP gene mutations play an important role in thepathogenesis of leukemia. After SHIP knockout mice, bone marrow cells werehighly proliferative, hepatosplenomegaly and infiltration of bone marrow cellsin vital organs,which are similar to human chronic myelogenous leukemia(CML) phenomenon[2]. Metzner et al[3]will SHIP gene transfect into CD34+cells of juvenile myelomonocytic leukemia (JMML) patients peripheral blood, found GM-CSF dependent proliferation and colony formation decreased,suggesting that SHIP may effectively block the precursor cells of JMML toGM-CSF hypersensitivity, which can be used for the treatment of JMML.Phosphatidylinositol3-kinase (PI3K)/Akt/mammalian target ofrapamycin (mTOR) signaling pathway (PI3K/Akt/mTOR) in maintaining thenormal function of cells play an important role, it is involved in cell growth,differentiation and metabolism process, is one of the important signalspathways in the body. The phosphatidylinositiol3-kinase (PI3K), AKT,mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival.The pathway can be abnormally activated in childhood acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (AML), and chronicmyelogenous leukemia (CML), as well as in some pediatric lymphomas andlymphoproliferative disorders. PI3K catalyzes the phosphorylation ofPhosphatidylinositol D-3,the PI-4-P and PI-4,5-P2were converted toPI-3,4-P2and PI-3,4,5-P3, PIP2and PIP3as a second messenger canactivate molecule protein kinase B (PKB/Akt) of downstream[4], and thusinvolved in cell proliferation, apoptosis and other processes. In acute leukemia,you can see the frequent activation of PI3K/Akt signaling pathway, PI3Kphosphorylate Ser473and Thr308of Akt, further activate many effectors ofdownstream[5,6]. Mammalian target of rapamycin (mammalian target ofrapamycin, mTOR) is one of the main molecular in the PI3K/Akt pathwaydownstream. mTOR belongs phosphoinositide3-kinase-related kinase family,the catalytic kinase domain of the carboxyl-terminal is highlyhomologous with catalytic domain of PI3K. mTOR through its downstreamtwo translation-related protein4E-BP1and p70S6K1(S6K1) play role.4E-BP1is Inhibitory factor of eukaryotic cell translation initiation factor4E(eIF4E), affect protein synthesis by binding to eIF4E. Thus, abnormalactivation of PI3K/Akt/mTOR signal transduction pathway may play animportant role in the development of leukemia and drug resistance, which isexpected to become effective target for treatment of leukemia. This study was to investigate expression of bone marrow leukemiccells SHIP and its downstream PI3K/Akt/mTOR pathway related molecules(eg:4E-BP1, p70S6K, HIF-1, VEGF) in patients with CML. And furtherresearch SHIP gene mechanisms in the pathogenesis of chronic myeloidleukemia and provide a scientific basis for the gene therapy of leukemia.Methods:1Collect of bone marrow specimens chronic myelogenous leukemiachronic phase and blastic phase patients, specimens are from chronicmyelogenous leukemia patients of the Second Hospital of Hebei MedicalUniversity in2012-2013,a total of27cases, the first experimental group ischronic myeloid leukemia in chronic period, a total of17cases, the secondexperimental group is chronic myeloid leukemia blastic phase, a total of10cases, all patients go through MICM that morphology (Morphology, M),immunology (Immunology, I), cytogenetics (Cytoge-netic, C), and moleculargenetics (Moleculargenetics, M) diagnosis, conform the diagnostic criteria ofFAB.10healthy volunteers as a control group, the experimental group of27cases, including15males and12females, age of21-67years, mean age44years, a total of10cases in the control group,5males and5females, age25-50years, mean age36years, all specimens extracted mononuclear cells,and saved-80°C refrigerator.2Realtime-PCR method to detect expression levels of intracellularSHIP and PI3K/Akt/mTOR signaling pathway related molecules (4E-BP1)mRNA;3Western Blot detect expression of SHIP and4E-BP1in protein levels4Analysis data by Statistics.Results:1The37cases of clinical specimens we collected showed the SHIP geneand4E-BP1gene were expressed at the protein and mRNA level.2Expression of SHIP gene mRNA Realtime-PCR results showed thatexpression levels of SHIP gene in chronic phase and blastic phase of chronicmyeloid leukemia lower than the control group(P <0.01), and the expression levels of SHIP in blastic phase lower than in chronic phase (P <0.01).3Expression of SHIP gene mRNA Realtime-PCR results showed thatexpression levels of4E-BP1gene in chronic phase and blastic phase ofchronic myeloid leukemia higher than the control group(P <0.01), and theexpression levels of4E-BP1in blastic phase higher than in chronic phase (P<0.01).4Expression of SHIP protein Western blot results showed thatexpression levels of SHIP protein in chronic phase and blastic phase ofchronic myeloid leukemia lower than the control group (P <0.01), and theexpression levels of SHIP protein in blastic phase lower than in chronic phase(P <0.01).5Expression of4E-BP1protein Western blot results showed thatexpression levels of4E-BP1protein in chronic phase and blastic phase ofchronic myeloid leukemia higher than the control group (P <0.01), and theexpression levels of4E-BP1protein in blastic phase higher than in chronicphase (P <0.01).Conclusion:1With the progression of chronic myelogenous leukemia, expressionlevels of SHIP genes mRNA showed a decreasing trend, consistent with theexpression level of protein. Considered the progress of chronic myelogenousleukemia related to SHIP gene.2With the progression of chronic myelogenous leukemia, expressionlevels of4E-BP1genes mRNA showed a decreasing trend, consistent with theexpression level of protein. Considered4E-BP1gene play an important role inthe progress of chronic myelogenous leukemia.