The Mechanism Research Of The Interaction Between Integrin β1 And TUBA1A In Cell Adhesion Mediated Drug Resistance(CAM-DR) Of Non-Hodgkin’s Lymphoma

ObjectiveCell adhesion mediated drug resistance(CAM-DR)is an important reason for incurable of non-Hodgkin’s Lymphoma(NHL).This study was aimed to verify the mechanisms and the significance of the interaction between integrin β1 and tubulin-alpha 1A(TUBA1A)in cell adhesion mediated drug resistance(CAM-DR)of non-Hodgkin’s lymphoma.Methods1.lymphoma cells were allowed to adhere to fibronectin(FN)or human bone marrow stromal cell line HS-5,Western blot assays were performed to analyze the expression of integrin β1 and TUBA1 A.Calein AM assays were performed to verify NHL cells adherent ability.CCK-8 assay and flow cytometer were performed to verify the role of knockdown of integrin β1 in cell apoptosis and CAM-DR in NHLs.2.HEK 293 T cells were transfected with HA-tagged integrin β1 and Flag-tagged TUBA1 A.CO-IP were demonstrated the interaction between integrin β1 and TUBA1 A.Then,construct the truncate plasmids of integrin β1,CO-IP were demonstrated the correct domain interacted with TUBA1 A.3.Lymphoma cells transfected with HA-tagged integrin β1 and(or)TUBA1A-siRNA.The interference efficiency was confirmed by western blot analyses.CO-IP were demonstrated the interaction between integrin β1and TUBA1 A,and Western blot were used to verify the changes of their interaction.Calein AM assays,CCK-8 assay and flow cytometer were performed to verify the role of the destruction of the interaction between integrin β1 and TUBA1 A in cell apoptosis and CAM-DR in NHLs.4.Lymphoma cells transfected with Flag-tagged TUBA1 A or TUBA1A-siRNA.Flow cytometer assays were used to verify the effect of TUBA1 A overexpression on membrane expression of integrin β1 and the activity of integrin pathway.Then,CCK-8 assay and flow cytometer were performed to verify the role of TUBA1 A knockdown in cell apoptosis and CAM-DR in NHLs.Results1.The expression of ITGB1 and TUBA1 A were increased after cells adhesion to FN or HS-5.As expected,cell adhesion rate were decreased after ITGB1 knockdown.CCK-8 assay and flow cytometer assays revealed that ITGB1 knockdown increased cells sensitive to doxorubicin.2.CO-IP assays demonstrated the interaction between integrin β1 and TUBA1 A were in both HEK-293 T cells and NHL cells,and the cytoplasm tail of integrin β1 interacted with TUBA1A3.In NHL cells,knockdown of TUBA1 A decreased the interaction between integrin β1 and TUBA1 A.Then,CaleinAM assays,CCK-8 assay and flow cytometer revealed that the drug protection mediated by integrin β1 were abrogated by TUBA1A-siRNA#3.4.Flow cytometer and Western blot results revealed that overexpression of TUBA1 A increased integrin β1 membrane clustering.Interestingly,TUBA1 A knockdown decreased the activity of integrin mediated single pathway.CCK-8 assay and flow cytometer revealed that TUBA1 A knockdown decreased cell viability and CAM-DR through blocking the activity of integrin pathway in NHL.Conclusions1.The expression of Integrin β1 and TUBA1 A may help to promote the adhesion of NHL cells to HS-5 cells,and then participate in regulation the process of cell adhesion mediated drug resistance;2.In the NHL cells,Integrin β1 interacted with TUBA1A;3.The effect of Integrin β1 in regulating cell adhesion,apoptosis and drug resistance can be reversed by TUBA1 A.4.TUBA1 A promotes Integrin β1 membrane clustering and CAM-DR through regulating activity of Integrin β1 and the downstream target gene in NHLs.