The Study Of The Regulation Of Myeloid Leukemia Cell Invasion By Eph/Ephrin

Background:With the discovery of new molecular target drug and development of hematopoietic stem cell transplantation, the treatment of myeloid leukemia has made great progress, but still the treatment of extramedullary infiltration was one of the most important problems. Newly diagnosed patients with extramedullary infiltration were not response to chemotherapy and often replased or refractory, extramedullary leukemia are often feeds for relapse after hematopoietic stem cell transplantation. There is no conventional means to perform comprehensive monitoring on extramedullary infiltration leukemia, patients with extramedullary infiltration were diagnosed after self-perceptible symptoms developed, then missed the best time to cure. Therefore, to in-depth study mechanisms on myeloid leukemia extramedullary infiltration to find related targeting molecules is of great significance for the early diagnosis and treatment of extramedullary leukemia.Contact inhibition of locomotion (CIL) refers to a phenomenon that changes its direction of motion when the cell collides with another cell. Studies suggest that the invasion of malignant cells was related with CIL abnormalities. Eph tyrosine kinase belonging to (Receptor Tyrosine Kinase, RTK) family of receptors, the binding of Eph with its ligand will lead both tyrosine phosphorylation which can occur simultaneously, then the bidirectional signal transmission will mediate regulation of cell morphology, adhesion and sports. The expression were related to drug resistance, metastasis and invasion-related. In solid tumors such as breast cancer, prostate cancer is more coverage, while the reports about acute leukemia is still rare. Eph receptor activation can promote and CIL between neighboring cells, leading to rejection of the cells and the cells go adhesion formation in the tissue boundary, the correct orientation of the axon play an important role in regulating tumor cells to invasive.Our previous proteomic study found that small G proteins may play an important role in chronic myeloid leukemia, and then found that the receptor tyrosine kinase receptor EphB4 could to regulate chronic myeloid leukemia the resistant to imatinib by small G proteins. So we are focusing on Eph receptor family kinase receptor and its ligand Ephrin tyrosine in research. primary myeloid leukemia cells and myeloid leukemia cell lines were conducted extensive screening for t mRNA levels 23 family members Eph and Ephrin receptor tyrosine kinases in the pre-experiment, then we found that the RNA expression levels of Eph and Ephrin were corelated with myeloid leukemia invasion. Some studies thought CIL is an important regulator of tumor cell invasive behavior, and may be by the regulation of Eph receptors. However, how does CIL regulate the process of invasion and what is the role of Eph in the myeloid leukemia invasion were still unknown. This is the purpose of our research.Objective:To study the mechanism of myeloid leukemia invasion by activation of Eph/Ephrin and to clarify how CIL regulate leukemia myeloid invasion by activation of Eph/Ephrin, the results will provide new targets for the theory study and clinical treatment for myeloid leukemia with extramedullary infiltration.Method:1. The mRNA and protein levels of 22 eph and ephrin receptor tyrosine kinase family members were detected in myeloid leukemia cell lines (K562, HL-60, U937) and normal donors by QT-PCR and Western-blot; then the mRNA and protein levels of EphA5 and EphB4 in 13 acute myeloid leukemia patients (including 5 patients with extramedullary infiltration) and 10 normal donors;2. The RNA interference technology was used to knocking-down the expression of EphB4 and EphA5 in K562 cell line respectively. Then a stable low-expression of EphB4 in K562 cell line (K562-EphB4-sh) and stable low-expression EphA5 in the K562 cell line (K562-EphA5-sh) were successfully built;3. Transwell invasion and migration assay were used to detect invasion and migaration in different myeloid leukemia cell lines K562, U937, HL60, K562-EphB4-sh and K562-EphA5-sh. Confocal Time-lapse maging combined with image analysis software Volocity software were used to quantitative analysis of CIL in myeloid leukemia cell lines K562-K562, HUVEC-HUVEC, K562-HUVEC and in K562-HUVE after EphB4 or EphA5 were knocked-down;4. Transwell invasion and migration assay were performed to detect the invasion between K562 cells and K562 cells co-cultured with HUVEC;5. Ephrin ligand stimulation experiments:Transwell invasion and migration assay were used to detect K562 invasion and migration upon the treatment of ephrin A1-Fc, ephrin B2-FC, Fc control on different time points; while its downstream signaling pathways RhoA/MMps pathway were detected by Western-blot; the quantitative analysis of CIL by Confocal Time-lapse maging image analysis software Volocity software were performed and the accompany changes in Racl/Cdc42 were using analisied by Western-blot; immunofluorescence were also performed to analisied the combination localization of Ephrin and Eph. The SEM was used for analysis of changes of cell morphology.Results:1. The expression of Eph family members in acute myeloid leukemia cell lines and primary myeloid leukemia cells:1) mRNA levels:Compared to cells from normal bone marrow, the expression of EphB4 and EphA5 in K562, HL60 and myeloid leukemia with extramedullary infiltration were both higher than (P<0.001、P<0.001; P<0.05、 P<0.05; P<0.001、P<0.001).2) EphB4, EphA5 protein expression levels in acute myeloid leukemia lines and primary myeloid leukemia cells:Compared to normal bone marrow cells, the expression of of EphB4, EphA5, EfnAl, EphB1 in acute myeloid leukemia cells were higher; Compared to acute myeloid leukemia without extramedullary infiltration, the expression of EphB4, EphA5 in acute myeloid leukemia with extramedullary infiltration were higher.3) In patients with acute myeloid leukemia:Compared to normal bone marrow cells, the expression of EphB4 and EphA5 in 13 cases of myeloid leukemia patients were higher (P<0.001; P=0.001); moreover, the expression of EphB4 and EphA5 in 5 cases with extramedullary infiltration were higher than 8 patients without extramedullary infiltration (P<0.001; P=0.001); however, there is no significant difference in between different leukemia subtypes (P> 0.05).2. The plasmid fragment were successfully inserted to K562 cells by sequencing validation, two stable low-expression of EphB4 K562 cell line and K562-EphB4-sh stable low-expression cell lines successfully built. The transfection were 80% and 90% respectively after lentiviral infection and flow cytometry sorting. The validation of EphB4 and EphA5 were performed by QT-PCR and Western-blot.3. EphB4/EprinB2 mediated myeloid leukemia cell invasion and migration:1) K562, U937, H1-60 cell migration and invasion comparison:Transwell migration assay showed that the cells migrated to chamber were different among K562, U937 cells and HL60 (F=310.011, P=0.000), moreover, invasion assay showed that the cells pass through matrigel were different among K562, U937 cells and HL60 (F=295.360, P=0.000), furthermore, K562 and U937 could pass through matrigel, and the cells invaded to matrigel were higher than HL60 cells (P=0.000、 P=0.000).2) The migration and invasion between K562-EphB4-sh cells and K562-EphA5-sh cell:Compared to K562 cells, the migration of K562-EphB4-sh cell migration decreased by 0.5 fold (P=0.000) while the migration of K562-EphA5-sh did not significantly decrease(P=0.808); moreover, the invasion of K562-EphB4-sh cell decreased by 0.35 fold (P=0.002) while K562-EphA5-sh did not significantly decrease (P=0.815).3) The migration and invasion after K562 co-culture with HUVEC:after co-culture with HUVEC, the numbers of K562 cells migrating through transwell chamber were significantly enhanced (P=0.035), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (P= 0.040).4) Ephrin ligand stimulation experiments:after ephrinB2-Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46-fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber didn’t significantly increase compared to Fc proteins stimulation (P=0.411),and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinAl-Fc stimulation. Moreover, the associated signaling pathways: RhoA/MMps were found to increase after the treatment of Ephrin B2-Fc for 45 min. Additionally, the immunofluorescence showed:Ephrin direct conjunction with Eph, leading to K562 cell migration and invasion after the treatment of Ephrin Fc.4. EphA5/EprinAl mediated CIL in myeloid leukemia cells1) Quantitative analysis CIL between K562-K562, HUVEC-HUVEC ans K562-HUVEC:The homotypic CIL and the heterotypic CIL were determined by Time-lapse maging And confocal microscopy combined with image analysis software Volocity software along with quantitative analysis Cx. The results showed that The homotypic CIL existed between K562 and K562, HUVEC and HUVEC, while the heterotypic CIL existed between K562 and HUVEC.2) The CIL between K562-HUVEC after EphB4 or EphA5 were knocked down: after EphB4 or EphA5 were knocked down, the homotypic CIL between K562 and HUVEC were retored:the change of value Cx calculated by 23 pairs cells were significantly changed (P=0.000).3) Ephrin ligand stimulation experiments:the CIL between K562 cells and HUVEC did not significantly change after ephrinB2-Fc stimulation (P=0.892), however, the CIL between K562 cells and HUVEC significantly changed after EphrinAl-Fc stimulation (P=0.005). Meanwhlie, the associated signaling pathways: RhoA, Rac1, Cdc42 were found to increase after the treatment of Ephrin A1-Fc for 45 min.Conclusion:1. The high expression of EphB4 and EphA5 in myeloid leukemia cells were corelated with strong cell invasion; Once EphB4 or EphA5 were knocked down, the invasiveness of myeloid leukemia cell decreases;2. The homotypic CIL existed between K562 and K562, HUVEC and HUVEC, while the heterotypic CIL existed between K562 and HUVEC; Once EphB4 or EphA5 were knocked down, the homotypic CIL between K562 and HUVEC were retored;3. The activation of Eph/Ephrin will regulate myeloid leukemia cell invasion by two pathways:Activation EphB4/EprinB2 will activate the downstream signaling pathways RhoA/MMps, leading to matrix degradation to promote the invasion of myeloid leukemia cells; Activation EphA5/EprinAl will activate the downstream signaling pathways Racl/Cdc42, leading to heterotypic CIL damage to promote the invasion of myeloid leukemia cells.