The Regulation And Mechanism Of Matrix Stiffness On Epithelial-Mesenchymal Transition Of Osteosarcoma Cells Via MRTF-A

Objective1.To fabricate biocompatible polyacrylamide hydrogels with various stiffness and measure their mechanical properties;2.To investigate the effect of MRTF-A on epithelial-mesenchymal transition of osteosarcoma cells under different stiffness;3.To find the specific signaling pathway and mechanism that matrix stiffness regulates epithelial-mesenchymal transition of osteosarcoma cells via MRTF-A.Methods1.Polyacrylamide hydrogels with different stiffness were fabricated and tested by Instron universal testing machine;The CCK8 assay was performed to understand the effect of hydrogels on cell proliferation,and the establishment of hydrogel system were confirmed by observation of morphological changes of osteosarcoma cells under light/fluorescence microscope.2.Cell circularity was quantified by Calcein AM staining,the remodeling of cytoskeleton and nuclear translocation of MRTF-A were confirmed by immunofluorescence.Western blotting was used to determine MRTF-A and EMT protein expression in cells of different groups.The MRTF-A inhibitor-CCG203971 was used to intervene the translocation process to observe above effect.Cultureinsert assay and western blotting were performed to confirm the migration of osteosarcoma cells.3.The mechanism of matrix stiffness regulating MRTF-A signaling pathway,as well as the relationship between MRTF-A and classical TGF-? / Smad signaling pathway,were detected by Western blotting.Results1.Polyacrylamide hydrogel with various stiffness can be fabricated by different crosslink ratio of acrylamide and bis-acrylamide,which had no toxic effect on cells and suitable for cell growth.Cell morphology can be affected by matrix stiffness.The lower stiffness,the higher cell circularity.2.Actin polymerization and nuclear translocation of MRTF-A were regulated by matrix stiffness,which promoted EMT process and migration of osteosarcoma cells.In addition,the nuclear accumulation of MRTF-A was significantly inhibited by CCG203971,as well as the EMT process and migration effect.3.After activating FAK by matrix stiffness,the members of RhoA signaling pathway,ROCK and mDia,were up-regulated.Actin monomer could be polymerized to F-actin by mDia after separated from MRTF-A by ROCK,then remodeling the cytoskeleton.The EMT process was therefore promoted by the translocation of MRTF-A.Meanwhile,The classical TGF-? / Smad signaling pathway was not activated by matrix stiffness.ConclusionThe morphology of osteosarcoma cells can be affected by polyacrylamide hydrogel system with various stiffness.Matrix stiffness plays a significant role in the EMT process by reorganization of cytoskeleton and subcellular localization of MRTF-A in osteosarcoma cells.The EMT process can be inhibited by CCG203971.The mechanical signaling pathway plays an independent role in regulating EMT process.