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Background: Acute myeloid leukemia (AML) is a group of clinical and biological behavior of highly heterogeneous disease, AML incidence of uncontrolled myeloid leukemia cell proliferation, differentiation the obstacles, apoptosis blocked, but its specific etiology and The pathogenesis is not yet fully understood. With the continuous improvement of chemotherapy, AML complete remission (CR) rate of up to about 80%, but most patients will relapse, hematopoietic stem cell transplantation, there are still 30% of patients will relapse, treatment effects need to be further improved. Existing treatment modalities leukemia, chemotherapy after years of development and evolution, has been difficult to reassert improve efficacy, the development of new therapeutic strategies to improve the overall prognosis of leukemia, biological targeted therapy and immunotherapy treatment of AML research focus. Looking for new gene closely related AML incidence is expected to further improve the efficacy of the development of new targeted therapies. Brain and acute leukemia cytoplasmic (BAALC) gene as a new discovery, normal karyotype AML (NC-AML) patients with the most significant prognostic indicators, and one of the potential therapeutic targets, broad application prospects. BAALC gene is located on chromosome 8q22.3, full-length 89Kp, under normal circumstances, BAALC only expressed in neuroectodermal tissue of origin and the surface of the early hematopoietic cells, whereas in normal bone marrow and non-expression or low expression of the surface of mature peripheral blood cells. Its DNA sequence is highly conserved proteins encoded by no homology with other functional proteins currently known. Further research in leukemia BAALC expression with acute leukemia, especially AML close contact. The the Baldus study found that patients with high expression of the the BAALC gene in AML, ALL and CML blastic phase (CML-BP) CML in chronic phase (CML-CP) and chronic lymphocytic leukemia (CLL) expression, suggesting that BAALC gene may closely related AML and ALL. Bienz et al reported that 70% of patients with normal karyotype AMLN BAALC gene significantly high expression of BAALC expression levels and the prognosis of NC-AML, BALLC higher the expression levels of AML prognosis worse. The Baldus reported BAALC high expression of AML patients of their disease-free survival (DFS) and overall survival (OS) was significantly shortened. Multivariate analysis, show, BALLC independent of FLT-ITD, MLL-TD, one of the prognostic factors of the most significant in the NC-AML, compared with the low-expression cases, resistance rates, cumulative recurrence rate was significantly higher high risk of death 2.7 times. Our previous work detected by real-time quantitative PCR (RQ-PCR) BAALC gene expression in AML patients, also found that the majority of newly diagnosed AML can be BAALC high expression, while the control group were not detected BAALC expression, normal karyotype early governance AML high expression the BAALC by chemotherapy, the CR rate was significantly lower than those with low expression, OS shorter, the prognosis is worse. Since the the BAALC and AL research is still in its infancy, on the function of the the BAALC gene and protein, BAALC AL onset and refractory relapse in the role of the molecular mechanisms on the downstream transcriptional regulatory networks and their signal transduction pathways is not yet clear, whether through The antileukemic effect inhibit the expression of BAALC play no relevant literature, both at home and abroad. Over the years, the study of gene function is mainly dependent on the knockout and antisense nucleic acid technology, gene knockout there is a complex operation, long life cycle, and the antisense technique specificity is not strong, the efficiency is not high, are not suitable for The large-scale study of gene function. Discovered in recent years some of the small double-stranded RNA can be efficient, specific blocking of certain genes resulting in mRNA degradation in vivo, to induce the cells exhibited a specific gene deletion phenotype, called RNA interference (RNAi). RNAi main advantage is the reverse gene analysis, namely, the specific gene sequence is known, by inhibiting the expression of this specific gene function and individual phenotypic changes, and then get the corresponding contact of the gene and its functions. In addition, this technique also has a specific, efficient, fast and the target RNA cleavage sites for a certainty, high stability, and high penetration characteristics. Due to the powerful means of RNAi technology simple operation and suppression of target gene expression efficiency makes it the study of gene function. Objective: BAALC gene expression silencing by RNA interference (RNAi), the establishment of a stable and silent AML cell lines to study of BAALC gene in the pathogenesis of acute myeloid leukemia. Methods: 1. Targeting Synthesis interference BAALC gene expression oligonucleotide, annealing short hairpin RNAi expression vector digested connected to transform E. coli DH-5α, blue-white screening positive clones, plasmid DNA was extracted for agarose gel electrophoresis and sequenced to verify that the recombinant plasmid vector sequences constructed targeting BAALC RNAi expression vector. Recombinant vector expression of BAALC gene to liposomal transfection method import the AML cell line Kasumi-1 cells and KG1a cells, fluorescence microscopy of GFP expression by flow cytometry (FACS) to detect the transfection efficiency, G418 positive clones were screened cell lines, total RNA was extracted and total protein, RT-PCR and Western blot analysis, verify the effect of the interference, stable and silent cell lines. Statistical analysis: SPSS 13.0 software package processing, P ≤ 0.05 indicates significant difference. Results: 1, annealed shRNA Description Oligo single-strand annealing single electrophoretic bands visible by agarose gel electrophoresis 100bp at success. Take pGPU6/GFP/Neo empty plasmid with BamH Ⅰ, Bbs Ⅰ restriction enzyme digested overnight at 37 ° C was digested, the digestion product line agarose gel electrophoresis, visible 5kb at 57bp at two electrophoresis strip, plasmid extraction successfully digested purified by plastic cutting can be used for the next step ligation reaction. Can be used for subsequent connections to the carrier. Purified product is connected with annealing product transformation transformed E. coli DH-5α, blue-white screening positive clones, plasmid DNA was extracted again enzyme electrophoresis and DNA sequencing to verify the recombinant plasmid vector sequence is correct, BAALC successfully build targeted RNAi expression vector. 2 cells 48h after transfection observed by fluorescence microscopy of GFP expression, some cells were GFP positive transfected recombinant shRNA expression vector successfully into human myeloid leukemia cell lines. Transfection 48h the cells were collected, washed in PBS to the expression of GFP was detected by flow cytometry (FACS), and calculating the efficiency of transfection. Effect of transfection transfection group the 4μl and 2μl found the 2μl transfected group expression of GFP-positive rate (5%) the higher than 4μl transfected group (3%), 2μl group transfection better, follow-up experiments take optimization the 2μl system for transfection. Before and after RNA interference, the extraction of the total cellular RNA for semi-quantitative RT-PCR validation interference effects found after interference control GAPDH gene transcription level than before interference significantly with lowered, the experimental group cells in BAALC genes at the transcriptional level but also by varying degrees inhibition, compared with the control group, the experimental group BAALC gene mRNA transcript levels decreased by more than 60 to 70%. The description of the recombinant plasmid vector is smoothly introduced into the body of the leukemia cell lines, expression at the transcriptional level, smooth, and combined with the target sequence, the occurrence of RNAi role. Before and after RNA interference, total protein was extracted for Western blot experiments verify the moderating effect of the protein level, semi-quantitative analysis of the Band Scan image analysis software absorbance bands. The Westernblot results in RNA interference, GAPDH protein was significantly reduced, BAALC protein expression in the cells of the experimental group was significantly suppressed, from about 40 to 60%. Tip the recombinant vector RNAi can give full play the role of down BAALC expression. The interference effect there may be differences of the cell series. Conclusion: We successfully constructed siRNA expression vector interference BAALC gene targeting, liposomal transfection methods can successfully imported human AML cell lines, stable silent BAALC gene expression in human AML cell lines by G418 to 2 interference sites down BAALC gene expression was most obvious. Choose two sites stably down BAALC gene expression in the cell lines of human AML cell proliferation, differentiation and apoptosis.
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