Effect Of Integrinα5β1 On Adhesion And Invasion Of A549 Lung Adenocarcinoma Cells Induced By EGF

Objective To identify whether the expression of integrinα5β1 of A549 lung adenocarcinoma cells increase by stimulation of EGF, and determine increasing of adhesive ability and invasive ability mediated by EGF can be removed or eliminated through blocking integrinα5β1,and to further provide scientific proofs for metastasis mechanism of non small cell lung cancer and looking for new targets for treatment. Methods RT-PCR method and flow cytometry were employed to respectively determine the changes and possible change tendency at mRNA and protein level of integrinα5 and integrinβ1 of A549 lung adenocarcinoma cells under stimulation of (EGF) for 48 hours. Fn adhension assay and transwell chamber method were respectively employed to determine whether the adhesive and invasive ability of A549 lung adenocarcinoma cells changed correspondingly under the stimulation of EGF with different concentrations, and further determine whether the acceleration of adhesive and invasive ability of A549 lung adenocarcinoma cells can be counteracted by anti-integrinα5β1 monoclonal antibody. Results 1.RT-PCR: There was no significant difference in expression of integrinα5 mRNA among 3 different groups(p>0.05), but expression of integrinβ1 mRNA in 1ng/ml EGF group and 10ng/ml EGF group were both significantly higher than that of control group(p<0.05).However, expression of integrinβ1 mRNA between 1ng/ml EGF group and 10ng/ml EGF group was not significantly different(P>0.05).2. Flow cytometry:Expression of integrinα5 and integrinβ1 expressed on the surface of A549 cells are both positive, and expression of integrinα5 is higher than that of integrinβ1; There was no significant difference of expression of integrin alpha5 on A549 cells among 3 groups(p>0.05); The expression of integrinβ1 expressed on the surface of A549 cells in 1ng/mlEGF group and 10ng/mlEGF group were significantly higher than that of control group(p<0.01), but difference of expression of integrinβ1 on A549 cells between 1ng/ml EGF group and 10ng/ml EGF group were not(p>0.05). 3. Fn adhesion assay: Fn adhesion rate of cells in control group was significantly lower than that of 1ng/ml EGFgroup and 10ng/ml EGF group(p<0.01), but there was no significant difference in Fn adhesion rate between 1ng/ml EGFgroup and 10ng/ml EGF group(p>0.05). 4. Invision assay: Number of cells passed through membrane in control group was significantly lower than that of 1ng/ml EGF group and 10ng/ml EGF group(p<0.01), but there was no significant difference in number of cells passed through membrane between 1ng/ml EGFgroup and 10ng/ml EGF group(p>0.05). 5.Antibody blocking assay:Of 4 groups of cells,Fn adhesion rates between any 2 groups were sinignificantly different (p>0.05),and Fn adhesion rates of cells among 3 antibody blocking groups were negatively correlated with antibody concentrations;Of the 4 groups of cells,number of cells passed through membrane between any two groups were significantly different,but 1μg/ml anti-integrinα5β1 mAb group and 10μg/ml anti-integrinα5β1 mAb group were on exception(p>0.05).Conclusions:1.EGF can significantly increase the expression level of integrinβ1 of A549 cells at mRNA and protein level ,but the expression of integrinβ1 is not related with EGF concentration ,but the expression of integrinα5 can't be affected by EGF.2. EGF can significantly increase adhesive ability and invasive ability of A549,but don't continuously increase with the increasing of EGF concentration;3.Anti-integrinα5β1mAb can significantly attenuate the accumulation of adhesive ability and invasive ability of A549 cells induced by EGF.4.Intergrinα5β1 is hopeful to be a new target in the targeted treatment of non small cell lung cancer .