The Investigation Of CrkL Mediating The Malignant Behaviors Of Chronic Myeloid Leukemia Cell Line K562 And Corresponding Molecular Mechanism

Back ground: Chronic myeloid leukemia（CML） is a kind of clonal myeloproliferative disease. CML was the first disease with the unique chromosomal translocation, the so called ‘‘Philadelphia chromosome’’ t（9; 22）. At the genetic level, the translocation leads to the fusion of the breakpoint cluster region（BCR） gene and Abelson tyrosine kinase（ABL）. The 210-kDa protein encoded by BCR-ABL fusion gene achieved the constitutively elevated tyrosine kinase activity which results in a variety of biochemical changes in leukemic stem cells（LSCs） significantly, inducing the growth factor–independent proliferation, the inhibition of apoptosis, and the alteration of adhesive properties. Bcr-Abl activates a large amount of signaling pathways which lead to the uncontrolled proliferation, the inhibition of apoptosis, and the block of myeloid differentiation. Among these, many of these are thought to be a redundant fashion, only a few signaling has been reported to be critical for the Bcr-Abl-mediated oncogenic transformation. All of the transforming functions of BCR- ABL are dependent on its tyrosine kinase activity.CrkL（Crk-like） is a member of the Crk family which is a kind of adapter proteins and contain an NH2-terminal S H2 domain and two SH3 domains, CrkL mediated the formation of protein complexes which is required for the signal transduction in some biological processes, containing the cell survival, proliferation, adhesion, and migration. The difference in the structure between CrkL and Crk results in the strong preference of BCR- ABL for CrkL. CrkL is required for mediating the aberrant activity of BCR- ABL, and is constitutively phosphorylated in human CML cells which have been used as a marker of BCR- ABL kinase activity. CrkL, as an intracellular signaling adaptor protein, integrates and coordinates many biologically key signals in the cell.Objective: 1. To construct pGPU6/GFP/Neo-shRNA-CrkL expression vectors and abtain monoclonal shRNA-Crk L-K562 cell with CrkL down-regulated stably; 2. To investigate the influence of CrkL downregulation on proliferation, colony formation, migration, invasion, apoptosis, imatinib resistance properties of K562 cell in vitro; 3.To investigate CrkL mediating signal pathways on CML.Methods: 1. The CrkL siRNAs were designed according to the m RNA sequence of CrkL（NM₀05207.3） by the siDirect and whitehead software and a non-targeting sequence was designed as a negative control. The K562 cells were transfected with pGPU6/GFP/Neo-shRNA-CrkL and pGP U6/GFP/Neo-shRNA-NC expression vectors using Lipofactamine 2000 transfection agent. The monoclonal K562 cells with stable knockdown of CrkL were screened by the G418 selection and the limited dilution. The expression levels of CrkL in monoclonal shRNA-CrkL- K562（1）, shRNA-CrkL- K562（2） and shRNA-NC- K562 cell lines were confirmed by Western blot and qRT-PCR. 2. The changes in morphology and submicrostructure of corresponding K562 cells with CrkL down-regulation were observed by using transmission electron microscopy. 3. Trypan blue excluding assay was measured to test the influence of CrkL downregulation on the proliferation of K562 cells. 4. Colony formatting capacity was determined to investigate the CrkL knock-down on the cell growth ability of K562. 5. The transwell assay was determined to investigate the CrkL knock-down on the cell migration and invasion ability of K562. 6. Flow cytometry was determined to further investigate the CrkL knock-down on the cell cycle of K562 cells. 7. The Hoechst 33258 stain assay was determined to investigate the CrkL knock-down on the cell apoptos is and imatinib resistance of K562. 8. qRT-PCR and Western Blot were determined to investigate the CrkL mediating pathways in K562 cells.Results: 1.The pGPU6/GFP/Neo-shRNA-CrkL- K562（1）, shRNA-CrkL-K562（2） and shRNA-NC- K562 expression vectors were constructed successfully; 2. Monoclonal K562 cell lines with CrkL stable knockdown were obtained. A signif icant reduction in Crk L m RNA and protein levels was observed in shRNA-CrkL- K562（1） and shRNA-CrkL- K562（2） when compared with shRNA-NC- K562 cells. 3. Following CrkL down-regulation, the mitochondria in shRNA-CrkL- K562 cells（1） and shRNA-CrkL- K562 cells（2） were damaged seriously and mitochondria were obvious swollen, and in many mitochondria the cristae were almost perfectly disappeared, and many vacuolar mitochondria were observed. 4. Proliferation was significantly reduced in shRNA-CrkL-K562（1） and shRNA-CrkL-K562（2） cells when compared with shRNA-NC- K562 cells at 48, 72, 96 h.; 5. The number of colonies in shRNA-CrkL- K562（1） cells and shRNA-CrkL- K562（2） cells was observed a signif icant decrease when compared with shRNA-NC- K562 cells and the colonies size of shRNA-CrkL- K562（1） cells and shRNA-CrkL- K562（2） cells is also significant ly inhibited when compared with shRNA-NC-K562 cells; 6. The cell migration and invasion ability in shRNA-CrkL- K562（1） cells and shRNA-CrkL- K562（2） cells was significant decrease when compared with shRNA-NC- K562 cells; 7. A significant accumulation of the shRNA-CrkL- K562（1） cells and the shRNA-CrkL- K562（2） cells at S phase of the cell cycle when compared with shRNA-NC- K562 cells; 8. The number of apoptosis cells by Hoechst 33258 staining in the shRNA-CrkL-K562（1） cells and shRNA-CrkL- K562（2） cells are increased when compared with the shRNA-NC-K562 cells with 0.01μM, 0.1μM, 0.5μM of imatinib mesylate incubated 24 h.; 9. In the shRNA-CrkL-K562（1） cells and shRNA-CrkL- K562（2） cells inhibition of CrkL induces the megakaryocytic differentiation 10.Inhibition of CrkL induces the downregulation of PI3K- Akt/mTOR pathway, MAPK pathway, FAK/Src pathway, CrkL-STAT5 pathway p130CAS/CrkL/Dock180/RAC1 pathway and further decreasing the proliferation, clonogenicity, migration and invas ion, cell cycle and imatinib resistance in K562 cells.Conclusion: 1. The pGPU6/GFP/Neo-shRNA- CrkL and negative control expression vector were constructed successfully; 2. The monoclonal K562 lines with stable knockdown of CrkL were obtained; 3. CrkL downregulation decrease the proliferation, clonogenicity, migration and invasion, cell cycle and imatinib resistance and induce megakaryocytic differentiation in K562 cells; 3. Inhibition of Crk L induces the downregulation of PI3K- Akt/mTOR pathway, MAPK pathway, FAK/Src pathway, CrkL-STAT5 pathway and p130CAS/CrkL/Dock180/RAC1 pathway to inhibit the transformation of CML. 4. CrkL play a key role in CML malignant behavior and has a potential use as a novel biomarker for the therapy of CML.