The Role Of Rho In Stromal Cell-derived Factor-1-induced Jurkat Cell Migration

The migration of leukemia cells is a key step in the invasion.Stromal cell-derived factor-1(SDF-1)can induce cell migration by triggering the related signal pathways.The process of cell migration is closely related to the change of cytoskeleton,and RhoGTPase family mainly regulate the actin cytoskeleton.Rho is a mainly member of RhoGTPase family.Therefore,the role of Rho in SDF-1-induced migration of leukemia cells was researched in this paper.Firstly,Jurkat cells were treated with different concentrations of SDF-1(0,25,50,100 and 200 ng/ml)and the migration was observed by Transwell assay.Then,the Jurkat cells were treated with the inhibitor of cytoskeleton and the connection between cell migration and cytoskeleton was examined.Moreover,the changes of cytoskeleton of Jurkat cells by SDF-1 stimulation were observed by confocal microscopy and detected by flow cytometry.ROS and NO are the intracellular key signal-effector molecules and we researched the role of them in SDF-1-induced cell migration.The production of ROS was detected by flow cytometry and the production of NO was detected by microplate reader.We researched the effects of ROS and NO on SDF-1-induced cytoskeletal changes and cell migration.Further,we treated Jurkat cells using Rho inhibitor and examined the connection between ROS/NO production and Rho by SDF-1 stimulation.Then,we studied whether Rho was involved in SDF-1-induced cytoskeletal changes and migration.The activation of RhoA,RhoB and RhoC by SDF-1 stimulation was detected by Western Blot.Finally,siRNA transfection was used to interfere the expression of RhoA and RhoC and we researched the effects of RhoA and RhoC on SDF-1-induced cell migration by Transwell assay.To study the role of RhoA and RhoC in SDF-1-induced migration of Jurkat cells,we interfered the expression of RhoGDI2 which is the inhibitory factor of RhoA and RhoC.Jurkat cells were then infected and observed under microscope to analyze the infection efficiency,and Western blot assay was conducted to detect the expression of RhoGDI2.Results showed that:1.SDF-1 induced cell migration by regulating rearrangement and polymerization of cytoskeletonSDF-1 induced the migration of Jurkat cells with the concentration-dependent-effect and the migration was more obvious with the increase of concentration.Jurkat cells were pre-treated with cytochalasin B,the cell migration significantly inhibited after SDF-1 stimulation for 4 h.The cytoskeleton made rearrangement and polymerization by SDF-1 stimulation.2.ROS and NO were involved in SDF-1-induced cell migration by regulating rearrangement and polymerization of cytoskeletonSDF-1 stimulated Jurkat cells for 0,1,2,3 and 4 h,the expression levels of intracellular ROS and NO at 4 h were significantly higher than the control group.Jurkat cells were pre-treated with NAC and L-NMMA,stimulated by SDF-1 for 4 h,the cytoskeletal rearrangement and polymerization and cell migration were significantly inhibited by SDF-1 stimulation.3.RhoA and RhoC were involved in SDF-1-induced cell migration by regulating rearrangement and polymerization of cytoskeletonJurkat cells were pre-treated with CT04,the expression of ROS and NO levels,the cytoskeletal rearrangement and polymerization and cell migration were significantly inhibited after SDF-1 stimulation for 4 h.RhoA and RhoC,rather than RhoB were expressed in Jurkat cells.SDF-1 induced RhoA and RhoC activation,while RhoA and RhoC were involved in SDF-1-induced migration of Jurkat cells.The RhoGDI2 shRNA lentivirus expression plasmids were constructed,packaged and infected Jurkat cells.RhoGDI2 sh1 and sh2 could significantly reduce the expression of RhoGDI2 in Jurkat cells.Conclusions:1.SDF-1 could induce the migration and the cytoskeletal rearrangement and polymerization of Jurkat cells,while SDF-1 induced cell migration of Jurkat cells by regulating cytoskeletal rearrangement and polymerization.2.SDF-1 could induce the production of ROS and NO in Jurkat cells,while ROS and NO were involved in SDF-1-induced cell migration by regulating cytoskeletal rearrangement and polymerization.3.SDF-1 induced the production of ROS and NO,which were dependent on the activation of Rho.SDF-1 induced the activation of RhoA and RhoC,as well as RhoA and Rho C were involved in SDF-1-induced cell migration by regulating cytoskeletal rearrangement and polymerization.The lentiviral plasmids encoding RhoGDI2 sh RNA which is the inhibitory factor of RhoA and RhoC had successfully infected Jurkat cells and interfered the expression of RhoGDI2Our research focused on the role of Rho in the migration of Jurkat cells induced by SDF-1,and elucidated the related signaling pathways in this process.This will provide a reliable theoretical basis for studying the migration mechanisms of SDF-1-induced acute lymphoblastic leukemia cells and offer a new therapeutic target for leukemia.