Establishment Of Overexpression Of BAALC Gene In Human Acute Leukemia Cell And The Research On Its Biological Activity

BackgroundAcute leukemia (AL) is a heterogeneous group of diseases characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells as well as blockage of myeloid differentiation at different stages. With the continuous improvement of chemotherapy, AML in complete remission (CR) rate of 70%, but most patients relapse, even if hematopoietic stem cell transplantation 30% of patients still relapse, Refractory or/and relapse lacking effective treatment was still the most arduous problem in leukemia, which may benefit from more intensive consolidation chemotherapy regimens and allogeneic hematopoietic stem cell transplantation (allo-HSCT).Brain And Acute Leukemia, Cytoplasmic (BAALC), located on chromosome 8q22.3, as one of the most important independent risk indicators within the heterogeneous group of cytogenetically normal AML patients, predicted high minimal residual disease and inferior survival. Baldus et al found that high BAALC was expressed in subsets patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML) in blast crisis, whereas no BAALC expression could be detected in patients with chronic-phase CML or chronic lymphocytic leukemia (CLL). Given the fact that BAALC expression in normal bone marrow is restricted to the compartment of progenitor cells and that it shows high expression in a subset of leukemic blasts, BAALC may be seen as a stage-specific marker regulated during hematopoiesis and aberrantly expressed in leukemogenesis. The function of the BAALC protein in hematopoiesis and leukemogenesis contributing to a more aggressive behavior of AML has yet to be identified.Bienz reported in 70% of patients BAALC exist significantly high expression of genes and expression BAALC with NC-AML prognosis are closely related, BALLC higher expression, AML patients with the worse prognosis. Baldus also reported high BAALC expression in conjunction with FLT3 mutation status successfully identifies high-risk patients within the heterogeneous group of cytogenetically normal AML patients. High BAALC expression is established as one of the most important independent risk factors associated with resistant disease, a high CIR, and inferior survival, disease-free survival (DFS) and overall survival (OS) was significantly shorter. High BAALC expression remained a significant adverse prognostic factor for FLT3-ITD WT and MLL-TD patients, compared to those with low BAALC expression (hazard ratio 2.7), even when the 8 additional patients harboring a FLT3/ITD genotype with normal cytogenetics and treated on CALGB 9621 were included in the multivariable analyses for OS and DFS.Our Preparatory work by using the real-time quantitative PCR (RQ-PCR) to detect AML patients BAALC gene expression, also found that the majority of newly diagnosed AML can occur BAALC high expression, while the normal control group were not detected, CR rate was significantly lower of high BAALC expression AML than those with low expression, OS shorter suggesting it might develop into a risk stratification of AML new target. ObjectiveWe construct pMSCV-BAALC through the genetic engineering technology in vitro, and induct the expression vector into the homo leukemia cell lines. When the stable expression the BAALC gene homo leukemia cell lines was established, we can study the biological function of BAALC gene in the homo leukemia cell lines.Methods1.We withdraws mRNA from the KASUMI-1 cell line, and increases BAALC1-6-8 using the RT-PCR technology to obtain the goal gene fragment. Then we selects pMD 18-T Vector to make the TA clone. We use double enzyme and T4 ligase to make BAALC gene into the pcDNA3.1/myc-His vector, and then into the pMSCV vector to construct pMSCV-BAALC expression vector.2. The pMSCV-BAALC expression vector was inducted into the homo leukemia cell line (HL60/ADM, Molt-4 and K562). We use puro to obtain the stable cell line. Then, RT-PCR and Western blot was used to confirm BAALC gene expression.3. We raise the HL60/ADM cell line which can expresses BAALC gene stably. Then, we use the cytomorphology, MTT and Flow Cytometry to examine the biological changes due to the overexpression of BAALC gene.4. The statistical analyses were performed with the statistical software package SPSS 13.0, and the significance was set at P values less than 0.05 in the our tests.Results1. We use the technology of digestion by double enzyme, RT-PCR and Sequencing to confirm that the BAALC gene was correctly insected into the pMSCV vector.2. After transfection 48 hours, we use the Flow Cytometry to examine the GFP expression, and calculate the best transfection efficiency.3. KASUMI-1 cell was selected as positive control. RT-PCR was used to determine the expressions of GAPDH, PGK and BAALC genes in untransfected and transfected pMSCV or pMSCV-BAALC vectors into HL60/ADM, Molt-4 and K562 cells. The results showed that, all samples can express GAPDH, the HL60/ADM, Molt-4 and K562 cells can not express PGK or BAALC genes, the HL60/ADM+ pMSCV, Molt-4+pMSCV and K562+pMSCV cells can express PGK gene, but can not express BAALC gene; the HL60/ADM+pMSCV-BAALC, Molt-4+ pMSCV-BAALC and K562+pMSCV-BAALC cells can express PGK and BAALC gene.4.Western blot was used to make sure the protein espression of BAALC gene. The results showed that the HL60/ADM, Molt-4 and K562 cells can not express BAALC protein, but the HL60/ADM+pMSCV-BAALC, Molt-4+pMSCV-BAALC and K562+pMSCV-BAALC cells can all express BAALC protein.5. Cytomorphology show that the percentage of polykaryocyte in HL60/ADM+ pMSCV-BAALC cell was more than that in HL60/ADM and HL60/ADM+pMSCV cells, which suggest that HL60/ADM+pMSCV-BAALC cell grow faster than HL60/ADM and HL60/ADM+pMSCV cells.6.MTT showed that the cell proliferation rate of HL60/ADM+pMSCV-BAALC cells was significantly faster than that in HL60/ADM and HL60/ADM+pMSCV cells(P=0.044 and 0.009).7. Annexin V/PI results showed that the apoptosis rate was significant lower than that in HL60/ADM and HL60/ADM+pMSCV cells(P=0.017 and 0.017).8.The cell cycle results showed that (G0+G1) phase and G2 phase in HL60/ADM+pMSCV-BAALC cells was more than those in HL60/ADM and HL60/ADM+pMSCV cells, and the S phase was much shorter than that in HL60/ADM and HL60/ADM+pMSCV cells. ConclusionThe pMSCV-BAALC vector was succesfully constructed and stable overexpression cell line was obtained. BAALC gene may increase the. cell proliferation of HL60/ADM.