The Construction Of Multiple Sirnas Eukaryotic Expression Vector For Bcr/abl Fusion Gene

Background:Chronic myeloid leukemia (CML) is a malignant clone of hematopoietic stem cell disease, which arises from the reciprocal translocation t (9; 22) defines as Philadelphia (Ph) chromosome. The molecular consequence of the t (9; 22) translocation is the creation of the fusion protein BCR-ABL, which is a constitutively active tyrosine kinase. Depending on the site of the breakpoint in the bcr gene, the fusion protein can vary in size. Nearly all patients with CML express a 210Kd BCR-ABL protein changing numerous intracellular signaling transduction pathways, regulated cell adhesion, proliferation and apoptosis, which lead to CML. Therefore, the search for effective suppression bcr/abl fusion gene, down the formation of p210BCR/ABL, or antagonize PTK activity of p210BCR/ABL and block p210BCR/ABL-mediated pathway, etc., have become a new strategy for treatment. Synthetic p210BCR/ABL PTK inhibitor (TKI) imatinib mesylate (Gleevec), could specifically block the tyrosine kinase activity.It has achieved good clinical efficacy in CML and brought a new era for the treatment of CML.However, Some patients can not tolerate the side effects occurred, and some emerge the drug resistant.The emergence of drug resistant caused by bcr/abl point mutations, bcr/abl gene amplification, bcr/abl over expression. Although second-generation tyrosine kinase inhibitor nilotinib, dasatinib can solve most of the drug resistance , the gradual emergence of drug resistance will inevitably.The role of target for TKI is the BCR/ABL fusion protein,which can not really solve the root cause——bcr / abl fusion gene and can not cure CML in theory.Thus an ideal molecular target for bcr/abl fusion gene of the treatment becomes a research hotspot.RNA interference (RNAi) has emerged as an emerging gene blocking technology in post-transcriptional level over the last decade. Compare with other blocking technology, such as gene knockout, antisense oligonucleotide technology, RNAi has more rapid, effective and sequence-specific advantages. Studies have shown that the siRNA specific for the bcr/abl mRNA can significantly reduced bcr/abl gene expression in K562 cell, inhibit cell proliferation and induce apoptosis. However, this chemical synthesis of siRNA can easily degrade, unstable and RNAi effect is short. Screening efficient siRNA and bring to be lasting stable expression, using the expression vector in mammalian cell to express hairpin siRNA, is a alternative method for chemical synthesis in vitro. RNAi caused by this method is closer to the natural mechanism and may be used in gene therapy.In this study, We use the siRNA eukaryotic expression vector containing 1～6 units which has built. We first design and build a single siRNA eukaryotic expression vector for bcr/abl fusion gene, and then transfected K562 cell; detect the level of bcr/abl mRNA, BCR/ABL protein to confirm RNAi effect for the siRNA. On the basis of this study, we design and build the eukaryotic expression vector of multiple siRNAs for bcr/abl fusion gene to maximize the RNA interference effect, which provide a tool to further explore the gene therapy of CML. Objective:To construct multiple siRNAs eukaryotic expression vector for bcr/abl fusion gene, then provide a technological means to study siRNA in vivo and a new strategy to gene therapy research for CML.Methods:Part one: The construction and identification of the single siRNA eukaryotic expression vector for bcr / abl fusion gene1. Leukemia cell line K562 was grown in RPMI medium1640 supplement with 10% fetal calf serum in a humidified atmosphere containing 5% of CO2 at 37℃.2. The specific siRNA sequences for bcr/abl gene were designed according to some papers which had proven some effective siRNAs, the template sequences were cloned singlely into p1-siRNA plasmid, and then identify using restriction enzyme digestion, sequencing.3. The recombinant plasmids were transfected into K562 cell lines by liposome, the bcr/abl mRNA and BCR/ABL protein expression was analyzed by RT-PCR and Western Blot. The cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay. These would not only further validate the effectiveness of the selected siRNA sequence, but also show that the siRNA eukaryotic expression vector system we selected is effective.Part two: The construction of multiple siRNAs eukaryotic expression vector for bcr/abl fusion geneOn the basis of siRNA and single siRNA eukaryotic expression vector which have proven effectively above, many same or different siRNAs were cloned into the multiple siRNAs eukaryotic expression vector p6-siRNA, and then construct eukaryotic expression vector of multiple siRNAs specific for bcr/abl fusion gene.Results: 1. Through restriction enzyme digestion and sequence analysis, the template sequences of siRNA eukaryotic expression vector were the same as the designing sequences, that the constructed p1-siRNA1, p1-siRNA2 and p1-siRNAn plasmid were successful.2. The p1-siRNA1, p1-siRNA2, p1-siRNAn and GFP plasmid were transient transfected into K562 cell lines through the liposome LTX. The transfected cell can be identified by the green fluorescence under the fluorescent microscope.The transfection efficiency is about 30%.3. Compared the K562 cell transfected p1-siRNA1 with the one transfected non-specific plasmid p1-siRNAn: The bcr/abl mRNA was descendented 27.6%; the expression levels of BCR/ABL protein was decreased 30.7%; then compared the corresponding rate of cell proliferation and apoptosis rate which are 40.1% vs 16.5% and 9.65% vs 1.79%. Similarly, the result of a simple comparison of K562 cell transfected p1-siRNA2 and the one transfected non-specific plasmid p1-siRNAn shows that: bcr / abl mRNA levels decreased by 28.7%.These results prove that the two selected siRNA sequences and siRNA eukaryotic expression vector system really works, so we can continue to build a multiple siRNAs eukaryotic expression vector for bcr / abl fusion gene.4. Througn restriction enzyme digestion and sequence analysis, the template sequences of multipl siRNAs eukaryotic expression vector were the same as the designing sequences, that the constructed the multiple siRNAs eukaryotic expression vector for bcr/abl gene was successful.Conclusion:1. The constructed plasmid p1-siRNA1 or p1-siRNA2 was transient transfected into K562 cell lines through the liposome LTX,and then analyzed the results of RT-PCR, Western Blot, MTT and FACS, which demonstrated the specific plasmid for bcr/abl fusion gene had some interference effect to K562 cell lines.This expression vector can produce RNA interference effect within cells.2. We design to build a multiple siRNAs eukaryotic expression vector for bcr/abl fusion gene on the basis of the study mentioned above.Through sequence analysis, the template sequences of multiple siRNAs eukaryotic expression vector were the same as the designed sequences, that the constructed multiple siRNAs eukaryotic expression vector for bcr/abl fusion gene was successful, which can be used for the remainder of the experiment,and then provide a tool to further explore the gene therapy of CML.