NCK1 Promotes The Angiogenesis Of Cervical Squamous Carcinoma Via RAac1/PAK1/MMP2 Signal Pathway

Objectives: The study was to explore the roles of Nck1 in the angiogenesis of cervical squamous cell carcinoma(CSCC)and the related moleclular mechanism.Methods: The protein expression in CSCC samples and cancer cells was evaluated with immunohistochemisty and western blotting,respectively.The cancer microvessel density(MVD)was assayed with CD34 endothelial labeling and counted according to Weinner's standard.Protein mRNA level was detected with real time quantitative PCR.Nck1 gene up-regulation(SiHa-Nck1+)and knockdown(SiHa-Nck1-)in cancer cells was achieved by gene transfection and siRNA,respectively.Cellular supernatant protein level was measured with ELISA.Proliferation,migration and tube formation of the Human Umbilical Vein Endothelial cells(HUVECs)was evaluated by CCK-8 cell viability assay,transwell chamber assay and in vitro Matrigel tubulation assay,respectively.Results: Nck1 showed gradually increased level from normal cervical epithelia and low-grade cervical intraepithelial neoplasia(LGCIN)to high-grade cervical intraepithelial neoplasia(HGCIN).The expression level of Nck1 in CSCC was significantly higher than that of HGCIN and was associated with cancer MVD.The HUVECs treated with supernatant of SiHa-Nck1+ showed significantly increased ability of proliferation,migration and tube formation than the HUVECs treated with normal SiHa supernatant,respectively.Compared with the HUVECs treated with normal SiHa supernatant,the HUVECs treated with supernatant of SiHa-Nck1-showed significantly decreased ability of proliferation,migration and tube formation,respectively.Nck1 gene transfection and siRNA led to increase and decrease of Rac1-GTP ? p-PAK1 and MMP2 level in SiHa,respectively.Furthermore,pretreatment with the Rac1 inhibitor(NSC23766)significantly decreased the level of Rac1-GTP,p-PAK1 and MMP2 in the SiHa-Nck1+.PAK1 activation inhibited in SiHa-Nck1+,the MMP2 level was notably decreased.However,the level of Rac1-GTP was not significantly changed.The cancer cells with inhibition of Rac1 or PAK1 also showed an impaired ability of proliferation,migration and tube formation.Conclusions: Nck1 is highly expressed in CSCC and promotes the angiogenesis-inducing capacity.The molecular mechanism may be related to Nck1-mediated activation of the Rac1/PAK1/MMP2 signaling pathway.