The Investigation Of The Expression And The Function Of SHIP Gene In The Chronic Myelogenous Leukemia

Backgroud: Chronic myelogenous leukemia (CML) is a kind of acquired malignancy which is caused by clonal proliferation after mutation in multiple potential stem cells. CML happens at an incident of 1:100,000 globally, composed of 15%-20% of the leukemia in adults. In our county CML ranks as the third most prevelant leukemia at an incidence of 0.36 in 100,000, just behind the acute myelogenous leukemia and acute lymphocytic leukemia.In recent years, with the development of molecular biology and clinical medicine study, great progress has been made in the etiology study of CML. The discovery and study of Ph chromosome and fusion gene of bcr/abl contribute a lot to the diagnosis and therapy of CML. Bcr/abl is a negative regulator of apoptosis, it can increases the cells number by decreasing apoptosis. The increased instability of the genoome makes it easy for secondary mutation to happen in these kind of cells. It is still not very clear why bcr/abl causes malignant proliferation. SHIP protein is a downstream substrate of bcr/abl , it is exclusively expressed in hematopoietic cells, and was found as a negative regulator in PI3K/Akt pathway. Our study group has found out that mutations exist in the acute leukemia patients in our previous study. However, the function of SHIP in the development of CML especially in the clinical study still leaves us with unanswered questions. In our study we examined the expression of the SHIP gene in CML patients and investigated the function of SHIP in the development of CML. The study was carried out in four parts: 1. detect the expression of SHIP gene in CML patients through FQ-PCR; 2. Study the expression change in cell line K562 cells after transfecting the small interfere RNA specific to bcr/abl gene; 3. Use SHIP gene knockout bone marrow receptor mice to study the influence of SHIP gene to the function hematopoietic system; 4. Through examination of the change of p-Akt in PI3K/Akt pathway to study the mechanism of the influence of SHIP in apoptosis.Method:1. real time quantitative PCR was employed to test the expression of SHIP gene in the Chronic myelogenous leukemia.2. synthesis and transfection of siRNA specific bcr/abl fusion gene into K562 cells and then use FQ-PCR and western blot to test the expression of bcr/abl and SHIP at both gene and protein level.3. construct SHIP-/- bone marrow recipient mice to see the influence of SHIP on the hematopoietic system.4. study the p-Akt level change in the SHIP negative bone marrow cells through Western blot.Results:1. SHIP gene expression decreased in the CML patients.Results from the FQ-PCR revealed that the SHIP mRNA expression significantly lower in CML patients compare to the normal controls.2. the fusion gene of bcr/abl inhibit the expression of SHIP.2.1 increased SHIP mRNA was found in K562 cells after blocked the bcr/abl fusion gene.After block the expression of bcr/abl fusion gene in K562 cells with siRNA specific to bcr/abl, the expression of bcr/abl gene significantly blocked comparing to the blank and non-specific interfered controls. (p=0.0039, p=0.0039 respectively) While the expression of SHIP gene increased in the specific interfered group (P =0.037,P =0.016 respectively). The average values are:bcr/abl gene[specific interfered group (5.63±0.97)×105;blank :(27.14±3.05)×105 ; non specific siRNA tranfected group :(22.42±2.15)×105];no significant difference was seen between the later two groups ( P=0.055 ); SHIP gene specific siRNA transfected group (3.58±0.37)×105],blank [(1.52±0.37)×105],non-specific siRNA interfered group [(1.53±0.20)×105copy],no significant difference was seen between the later two groups(P =0.81)。 2.2 the expression of SHIP protein increased in K562 cells after blocking the expression of bcr/ablWestern blot results indicated that the expression of p210 (production of bcr/abl) greatly decreased after transfection with the bcr/abl siRNA in K562 cells compared to the no transfection control and non-functional bcr/abl siRNA group. No significant difference was found between the latter two groups. SHIP protein p145 can hardly be detected in the blank control and the non-functional bcr/abl siRNA group while in the bcr/abl siRNA interfered group the p145 significantly increased.3. infiltration of organs and hematopoietic changes happen in SHIP knockout bone marrow recipients3.1 SHIP -/- bone marrow recipients showed signs of infiltration just as in leukemia patientsThree months after transplantation, some of the SHIP knockout bone marrow recipients showed paralyzed rear legs. They could not bear their body weight, but reaction to pain was not different from the normal controls, which indicate the paralysis may be due to the damage of the motorneuron. This phenomenon mirrors what happens in leukemia patients. After dissection,enlarged lungs and spleens were found in the knockout bone marrow recipients compared to the normal controls.3.2 .SHIP knockout bone marrow recipients showed an increased ratio of neutrophils in peripheral blood.The peripheral blood of the SHIP knockout bone marrow recipients showed increased neutrophils by blood smear. However, no obvious primary cells or blast cells were found in the smears and the complete blood count(CBC). The CBC results showed equal amount of white blood cells, but the components (Neutrophils,Lymphocytes,Eosinophils) of the peripheral blood are significantly different between the two groups.3.3 Histology analysis revealed enlarged lungs and spleens were due to the infiltration of the myeloid cellsCompared with normal controls, SHIP-/- bone marrow recipients show significant pulmonary consolidation in some area of the lungs. Microscopic analysis revealed the consolidation due to the infiltration of the myeloid cells, mainly focused in the alveolus. Some neutrophils appear in the bronchus. The structure of the spleen was significantly damaged. The normal structure of the lymph follicles and the border of red pulp and white pulp disappeared due to the infiltration of vast amounts of myeloid cells.3.4 Morphorlogical study shows neutrophils increased in the SHIP knockout bone marrow recipeints spleen and peripheral blood.Compared with the normal control spleen, increased neutrophils were found in the knockout bone marrow recipients'spleen and peripheral blood but no obvious blast or primary cells are found in the peripheral blood.3.5 Immune analysis indicated the loss of SHIP in the bone marrow increased the percentage of neutrophils in the bone marrow spleen and peripheral blood.Stained the myeloid cells with Gr-1/Mac-1 antibody for flow cytometry analysis, we further analyzed the percentage of the myeloid cells in the previously described organs like bone marrow, spleen and peripheral blood. The results indicated that the percentage of myeloid cells in the SHIP negative bone marrow(Gr1+Mac1+)(40.38±0.87)% is nearly two times of that in the WT (20.91±1.11)% mice bone marrow. In the spleen the difference was even bigger, it was almost four times different from the normal controls.[ (14.47±0.67)% vs(3.49±0.47)%]. In peripheral blood in the SHIP negative bone marrow recipients the average number was (60.71±1.72)% while in the WT recipients it was only (23.38±0.62)%3.6 Colony assays indicate the loss of SHIP gene caused enhanced proliferation in myeloid cells.The colony numbers formed by the SHIP negative bone marrow cells are significantly larger than that formed by the WT bone marrow cells indicating that in the SHIP negative bone marrow there are higher ratios of progenitors or stem cells compared to wild type controls. This was also shown by CFU-MIX and CFU-gm assays. From the size of the colony we can also tell a higher proliferation ability of SHIP negative bone marrow cells.3.7 Migration Assay showed an enhanced migration ability of SHIP negative bone marrow cells.The migration test showed an enhanced moving ability of the SHIP negative bone marrow cells. The number of cells that migrated through the wells in the SHIP negative group(0.35x105/2x105) are significantly higher than those in the WT group(0.07x105/2x105 )(P<0.05).3.8 Cells from the SHIP negative bone marrow show reduced apoptosis induced by serum free starvation.4. Increased level of p-Akt was found in SHIP knockout bone marrow recipients.Results from the Western blot indicated that SHIP can significantly inhibit phosphorylation of Akt. The absence of SHIP causes the significant increase of the p-Akt, in bone marrow cells stimulated with GM-CSF.Conclusion:1. Lower expression of SHIP was found in CML patients2. Bcr/abl fusion gene inhibits the expression of SHIP gene.3. The loss of SHIP gene enhanced the proliferation and migration of hematopoietic cells, but inhibited apoptosis. Higher levels of p-AKT were correlated to increased sensitivity to GM-CSF in the KO cells.4. SHIP gene regulates hematopoiesis through the PI3K/Akt pathway.