ICAM-1 Regulating The Expression Of FAK And Its Function In Endothelial Cell

Inflammatory response is an important immune defense against the invasion of host by microbial pathogens.It is a host response that responds to tissue damage,initiates multi-factor chemical signals and continuous actionon diseased tissues.This involves the activation and directional migration of leukocytes(neutrophils,monocytes and eosinophils)from the venous system to the injury site.Intercellular adhesion molecule 1(ICAM-1)and focal adhesion kinase(FAK)play an important role in the activation and transendothelial migration of leukocytes.FAK and ICAM-1 regulate the activation of neutrophils.ICAM-1 interacts with ligands on activated leukocytes to activate the endothelium,thereby mediating leukocyte transendothelial migration.FAK affects the transendothelial migration process by regulating the flipping of the adhesion spot.Our laboratory has shown that ICAM-1 can regulate the expression of FAK through miR-15b-5p,but the specific pathway is still unclear.In order to better understand the coordination mechanism between ICAM-1 and FAK in endothelium,the mechanism of ICAM-1 regulating FAK expression mediated by miR-15b-5p and the effect of this signaling pathway on cell function were studied in this study.The main findings are as follows:1.ICAM-1 affects the expression of miR-15b-5p in endothelial cell:A length of 2049 bp DNA sequence of miR-15b-5p was inserted into the empty plasmid pGL3-Control vector to construct miR-15b-5p double enzyme reporter vector.Using 293 T cells as a model,a double fluorescence experiment was performed.The results showed that ICAM-1 affects the expression of miR-15b-5p in endothelial cell.2.ICAM-1 promotes the expression of FAK protein in endothelial cell:Immunofluorescence experiments were performed using b.End.3 cells as a research model.There are four groups in the experiment,which are(1)Basic;(2)ICAM-1;(3)ICAM-1+NC;(4)ICAM-1+siFAK.Compared with the control group,the expression of FAK protein was significantly increased by transfection of ICAM-1.Compared with transfected the ICAM-1 plasmid and control siRNA,the expression of FAK protein was significantly decreased when ICAM-1 and siFAK were simultaneously transfected.The results indicated that ICAM-1 positively regulates the expression of FAK.3.ICAM-1,MiR-15b-5P,FAK affect cell proliferation ability:The CCK-8 experiment was performed using b.End.3 cells as a research model.The experiment was divided into two parts.Firstly,the effects of ICAM-1 and FAK on cell proliferation ability were preliminarily explored.There were four groups in the experiment,namely(1)basic;(2)ICAM-1;(3)FAK;(4)NC;(5)siFAK;(6)ICAM-1+siFAK.The experimental results showed that ICAM-1 and FAK promote cell proliferation.Next,the effect of MiR-15b-5P on cell proliferation ability was investigated.The experiment consisted of four groups,namely(1)basic;(2)ICAM-1;(3)ICAM-1+miR-15b-5p mimics;(4)miR-NC;(5)miR-15b-5p mimics;(6)miR-15b-5p+siFAK.The experimental results indicated that miR-15b-5p inhibits cell proliferation.4.Knockdown of FAK to promote b.End.3 cell migration:To further explore the effect of this signaling pathway on cell function,b.End.3 cells were used as a model to transfect siFAK and cell scratch assays were performed.The experimental results showed that FAK knockdown can significantly enhance the migration ability of b.End.3 cells.5.The signaling pathway by which ICAM-1 regulates FAK expression via MiR-15b-5P is mainly present in endothelial cells:So far,the reported ICAM-1 induced signaling pathway is mainly present in endothelial cells.According to this,the scientific hypothesis is proposed: the signaling pathway is mainly present in endothelial cells,and three non-endothelial cells,A549,HepG2 and Hela,are selected.Two kinds of endothelial cells,b.End.3 and EA.hy926,were used as cell models for experiments.Overexpression and knockdown of ICAM-1 in non-endothelial cells A549,HepG2,Hela cells showed that the expression of FAK and miR-15b-5p was not significantly changed in cells;whereas in endothelial cells EA.hy926 and b.End.3 cells,overexpress and interfere the ICAM-1 showed significant changes in the expression of FAK and miR-15b-5p in cells.Therefore,this subject can conclude that miR-15b-5p mediates the signaling pathway of ICAM-1 regulating FAK expression mainly happened in endothelial cells.In conclusion,our results suggest that ICAM-1 can positively regulates FAK expression through miR-185b-5p in endothelial cells and promotes cell migration,thereby regulating the body's inflammatory response.