Study On EHD2-mediated Regulation Of Cellular Function Via EGFR

Objectives:EGFR Which possess tyrosine kinase activity is a transmembrane receptor,it takes important role in cell growth,differentiation and survival.EHD2 is a recently identificd protein which regulates Membrane trafficking.We have finded that down-regulation of EHD2 impaired the expression of EGFR of breast cancer,and cell growth and migration were also inhibited,but whether EHD2 regulation of EGFR transportion and signal trafficing is not clear:On the basis of using Hela cell line as model to investigate EHD2 regulation of EGFR,We will furth study the effect on cell proliferation,migration,transformation and the occurrence of tumor,and detecting the expression and location of EHD2 in breast cancer initially,so that we can investigate the mechanism of EHD2 and it’s regulation of EGFR in the occurrence and development of tumors.Methods:1.We used Western Blotting and immunofluorescence staining technology to detect the expression and location of endogenous EHD2 in Hela cells.2.We attenuate EHD2 expression in Hela cells,which was identified by Western Blotting analysis.Establishing stable knock-down cell colnes of Hela cells.3.Western Blotting analysis was used to detect the degradation of EGFR and the activation of EGFR and Akt.after knockdown EHD2 of Hela cells,which were stimulated by EGF for different times,so that we can futher identify the molecular mechanism of EHD2 regulating EGFR.4.Counting numbers,Chemotaxis assay and Soft Agar were performed to detect the role of EHD2 in cell proliferation,chemotaxis ability and clone growth.5.The SCID mice were adopted to investigate the effects of EHD2 knockdown in tumor growth of Hela cells.6.We used Western Blotting and Immunohistochemistry analysis to detect the expression of EHD2 in Breast cancer and coupled normal tissues.Results:1.Hela cells posses moderate level of endogenous EHD2,which is expressioned in nucleus,cytoplasm and cell membrane.2.After infection Hela cells,the expression of EHD2 significantly decreased,the expression of EGFR were decreased following with the down-regulation of EHD2.Stable clone of Hela were established.3.Western Blotting analysis revealed that down-regulation of EHD2 enhanced EGFR degradation and inhibited EGFR-Tyr1068 and Akt-Thr308 phosphorylation in Hela cells.4.Down-regulation of EHD2 impaired Hela cells proliferation(P<0.01),chemotaxis ability(P<0.01)and clone growth.5.SCID mice model revealed that EHD2 knockdown of Hela cells enhance the tumor growth.6.The expression of EHD2 in normal breast tissue is higher than breast cancer.Conclusions:1.Endogenous EHD2 regulated EGFR internalization negatively and suppressed the following degradation in Hela cells,so down-regulation of EHD2 enhanced EGFR degradation,decreasing the expression of EGFR.2.EHD2 took o role of promotting the cell function(such as proliferation and chemotaxis ability)by activating EGFR and AKT.3.EHD2 revealed inhibitting tumor in three dimensional.4.The role of EHD2 in the occurrence and development of tumors needs furth study.