The Molecular Mechanism Of Non-receptor Tyrosine Kinase Fyn Involved In Cervical Cancer Metastasis

Objectives We explore the effect of non-receptor tyrosine kinase Fyn on cervical cancer cell migration and invasion,and the details about regulating Furin by tyrosine kinase Fyn in human cervical cancer cells,providing new ideas for targeted therapy of cervical cancer metastasis.Methods Fyn and Furin protein level was analyzed by western blot,After Fyn was pretreated with gene silencing and overexpression system growth factor activation and Fyn kinase inhibitor.The changes in the migration and invasiveness Fyn si RNA transfected He La cells were observed using boyden migration and invasion assay.Furin enzyme activity were detected by enzyme activity assay.Co-Immunoprecipitation（Co-IP）and Dual immunofluorescence labeling and laser confocal microscopy（Confocal）assay were used to observe the interaction between Fyn and Furin.The effect of tyrosine phosphorylation of Furin was observed by Co-IP with anti-tyrosine phosphorylation antibody 4G10.The protein expressions of Furin and its substrate Menbrane type 1 matrix metalloproteinases（MT1-MMP）were detected after using activation or inhibition of Fyn protein by western blot.Results After Fyn protein expression was affected by gene delivery technology or chemical methods,Western Blot results showed that Fyn had no significant effect on Furin protein expression.We found that the activity of Furin enzyme did change significantly by PDGF and PP2 after 30 min pretreatment.Fyn si RNA transfected He La cells showed the significant lowered cell migration and invasiveness ability（P<0.05）compared with the control cells.Co-IP experiment showed that Furin was precipitated by Fyn,and there was interaction between Fyn and Furin in cervical cancer cells.Confocal assay showed that endogenous Fyn and Furin distributed in the thin layer of cell membrane and cytoplasm of He La,and overlapped.Immunoprecipitation of anti-tyrosine phosphorylated antibody4G10 showed that Furin was precipitated by 4G10 antibody.After the use of the Fyn inhibitor PP2,the binding ability of Furin to 4G10 was found to decrease with the dose of PP2.Western blot results indicated that pro-MT1-MMP was partially decomposed when Fyn was activated by Transforming growth factor-α（TGF-α）,MT1-MMP protein expression was increased.When Fyn was inhibited by PP2,the opposite result occurred.There was a significant pp56^Fyn expression induced by TGFαin the Golgi apparatus.Conclusions In cervical cancer cells,Fyn is involved in the metastasis of cervical cancer,possibly through the growth factor-Fyn-Furin signaling pathway.Figure11;Table2;Reference 111...