Study On The Correlation Between EphA4Receptor With Its Downstream Signaling Protein And Invasion Of K562Cell

Background and ObjectiveChronic myeloid leukemia is a malignant clonal proliferative disease originating from the pluripotent hematopoietic stem cells, and its basis of molecular biology is chromosome aberration and formation of abmormal gene fusion. Leukemia cells are characterized by abnormal proliferation and infiltration. Clinical manifestations of CML include splenomegaly, extreme elevation of peripheral white blood cells, increasing of immature granulocyte macrophage, alkali, eosinophilia, anemia and thrombocytosis. CML is divided into chronic phase, accelerate phase and blast crisis. Bone marrow primitive cell infiltration, such as skin or organ infiltration, and even the emergence of CNS leukemia can be seen in blast crisis.In recent years, the leukemia research launched by domestic and foreign scholars in basic and clinical aspects have made significant progress. New medicine of molecular target and improved chemotherapy regimens made tremendous progress in the treatment of leukemia, and allogeneic hematopoietic stem cell transplantation has greatly improved the survival rate of patients with leukemia. However, leukemic extramedullary infiltration is still a clinical problem to be solved urgently, chemotherapy in newly diagnosed patients with extramedullary infiltration is often poor and tend to relapse, and CML may become the root of relapse after hematopoietic stem cell transplantation. Now, there is no conventional means for extramedullary leukemic infiltration comprehensive monitoring. So the extramedullary leukemic infiltration mechanism should be studied in detail, and the exploration of related molecules and signaling pathway abnormalities, finding available targets are of practical significance for early diagnosis and treatment of extramedullary leukemia.Enhanced invision of leukemia cells is closely related to extramedullary infiltration.Invasion is an important biological characteristic of malignant tumors. Tumor cells of high invasion secret various dissolved enzymes which destruct extracellular matrix, at the same time, they also can be easier to spread to the surrounding tissue by the enhanced migration ability. As mentioned earlier, extramedullary infiltration occurs frequently in the blasting period, which relates to enhancement of invision.Cell migration can be adjusted by changing the binding density of receptors and ligands on celluar surface. The process includes the binding of celluar surface receptors and adjacent ligands, entrance of receptor-ligand binding activating regulation signals into cells, and cellular directional stretching by control signals’ remodeling cytoskeleton. It has been shown that integration signaling of Eph receptor family is the key point for regulating migration and invasion, and Eph receptor family is expected to become the breakthrough for the monitoring and treatment of extramedullary infiltration. Erythropoietin-producing human hepatocellular (Eph) receptor family is the largest subgroup in receptor tyrosine kinases(RTKs) with25members,including16Eph receptors:EphA (EphA1-EphA10)、EphB (EphBl-EphB6) and9Eph receptor ligands-ephrins:EphrinA (EphrinA1-EphrinA6)、 EphrinB(EphrinB1-EphrinB3). Each member of this family may play the role of ligand or receptor, Eph/ephrin is capable of binding a bidirectional signal. Eph receptor and ephrin ligand binding on different celluar surface can lead to tyrosine phosphorylation at the same time, which mediates bidirectional signal transmission and activates multiple downstream signaling pathways including Ras/Raf/MAPK and PI-3K/AKT signal transduction pathways, and these signaling pathways can’cross talk’ by each other. Among many of the downstream signaling proteins, the activation of Rho GTP enzyme can cause cytoskeletal remodeling and changes in cell adhesion, polarity and the direction of motion, which is an important regulator of cell migration. So far, studies on EphA4in extramedullary infiltration of CML have not been reported.EphA4receptor, the only receptor to combine with all nine ephrin ligands in Eph family, has diverse conformation. The feature of EphA4makes it capable of taking part in execution and regulation of biological function. Many studies have showed that as an important cell signal transduction protein, EphA4can adjust the cell morphology, and play an important role in cell migration, intracellular function, cell-matrix interaction, cell proliferation and apoptosis after activated by matching ligand. In tumor study, expression of EphA4is related to metastases and staging of gastric cancer. In neuropsychological research, escalation of EphA4will promote dendritic spine retraction and decrease its density by regulating cytoskeleton and morphology. Astin have found that in prostate cancer, prostate cancer cells with high expression of EphA activate EphA after contact with each other to promote spreading via mutual exclusion, at last, realize the cytoskeletal remodeling through Rho GTPase.Based on above researches, it can be hypothesized that EphA4and its downstream signaling proteins may have certain effects on the occurrence and development of CML, particularly on extramedullary infiltration which relatd with cell invasion force. It is a task that deserves research, and its significance lies in looking for treatment targets in the fight for extramedullary infiltration. K562cell comes from erythroleukemia acute thansformation of CML patients,it has the potential of angiogenesis and infiltration, so we choose the cell lines for research mechanism of EphA4in occurance and development of infiltrating and function in leukemia. We observe EphA4receptor expression level in myeloid leukemia generation cell,and then select K562as the research object. The expression level of EphA4mRNA and protein of K562are detected, then we created a EphA4over-expression K562stable cell lines and observed the changes of downstream signals proteins of EphA4.At the same time, the functional experiments confirm that the capacity of extramedullary infiltration is mainly caused by changes of invasion. From these observation, we will get knowledge about regulation of EphA4on the downstream signal of K562cell and its relationship with extramedullary infiltration, so that more effective target for extramedullary infiltration could be found, and also it is conducive to solve refractory and recurrent problems of extramedullary leukemia, with positive significance.Methods 1. EphA4protein expression level in bone marrow mononuclear cells of myeloid leukemia patients with extramedullary infiltration and without extramedullary infiltration were detected by Western Blot. EphA4mRNA and protein expression level of K562and of normal bone marrow mononuclear cells were detected by real-time quantitative PCR and Western Blot2.As the first part results showed the low abundance of EphA4mRNA level of K562cells, we creat K562stable cell lines of over-expression EphA4(K562-EphA4) by preparation restructuring cloning and sequencing、 building slow virus carrier、 packaging slow virus and transformation into the K562cells(K562-EphA4), then which will be used to study downstream signaling proteins and function of EphA4.3. The expressing level of phospho-RhoA and phosphor-Racl/cdc42protein were detected in K562and K562-EphA4by Wetern Blot in order to study the effect of EphA4on their activity.4. Compare invasion force by migration test and transwell invasion test to observe the capacity of extramedullary infiltration between K562and K562-EphA4cells.Results1. Western Blot results showed that the EphA4protein level in bone marrow mononuclear cells of myeloid leukemia patients with extramedullary infiltration was higher than that in myeloid leukemia patients without extramedullary infiltration,and the difference was statistically significant (P<0.05). qRT-PCR and Western Blot showed that EphA4mRNA and protein level in K562cells were higher than that in normal bone marrow mononuclear cells, and the differences were statistically significant (P<0.05).2. Western Blot showed that EphA4protein level in K562-EphA4cells was higher than that in K562cells, and the difference was statistically significant (P<0.05).It showed that we created successfully a stable cell line which over-expressed EphA4.3. Western Blot showed that phospho-RhoA protein level and phospho-Rac1/cdc42 protein level in K562-EphA4cell is higher than it in K562, the difference of both are statistically significant (P<0.05).4. Transwell invasion experiment showed that the number of cells which pene trated through matrigel hydrogel and microporous membrane in K562-EphA4is more than that in K562cells, and the difference is statistically significant (P<0.05).Conclusion1. The results indicated that EphA4receptor may play a role in regulating invasion force of leukemia cells.Combine with large numbers of documents,we consider that it will be meaningful by study the role in cell invasion of EphA4receptor.2. We created successfully a stable cell line which over-expressed EphA4(K562-EphA4) and make a foundation for studying the role of EphA4in leukemia progress.3. The results of this study showed that over-expression of EphA4in K562cell may up-regulat the activity of RhoA protein and Racl/cdc42protein level. RhoA, Rac1, cdc42may be its downstream signal proteins, and plays a regulatory role, we hypothesized that EphA4may affect the capacity of infiltration by regulating the activity of RhoA, Rac1/cdc42protein.4. Transwell invasion experiment showed that the migration and invasion ability of K562-EphA4cell were higher than that of K562cells, which indicates that over-expression of EphA4can enhance migration and invasion ability in K562cells.5. To sum up, we considerd that EphA4could enhance its invasion force to promote extramedullary infiltration by up-regulating activity of its downstream signals such as RhoA, Rac1, cdc42protein. Therefore, EphA4is expected to become the target of monitoring and managing extramedullary infiltration, which is of great theoretical significance and practical application value for the treatment of CML.