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  • br Fig ENO regulates EMT in

    2020-07-08


    Fig. 4. ENO1 regulates EMT in GC cells. (A) Western blot analysis of the E-cadherin and Vimentin expression in EPZ031686 stably transfected with control-shRNA (NC) or shRNA against ENO1 (KD). GAPDH as a loading control. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right).
    (B) Western blot analysis of the E-cadherin and Vimentin expression in cells stably transfected with empty vector (VEC) or plasmid encoding human ENO1 (ENO1). GAPDH as a loading control. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right). Statistical significance was determined by a two-tailed Student t-test. *P-values < 0.05, **P-values < 0.01.
    Considering that ENO1 regulates PI3K/AKT signaling pathway in several cancers (Song et al., 2014; Zhao et al., 2015; Fu et al., 2015) and the PI3K/AKT signaling pathway contributes to tumor cell proliferation and metastasis (Liang and Slingerland, 2003; Gonzalez and Medici, 2014), we thus asked whether the AKT signaling pathway is implicated in the ENO1-mediated gastric cancer cell proliferation and metastasis. In accordance with the previous studies (Song et al., 2014; Zhao et al., 2015; Fu et al., 2015), we found that knockdown of ENO1 significantly reduced the phosphorylation of AKT in gastric cancer cells while ec-topic ENO1 expression had the opposite effects (Fig. 5A and B). We also found that the enhanced proliferation and migration ability induced by ENO1 overexpression was remarkably impaired after incubation with Ly294002 in SGC7901 gastric cancer cells (Fig. 5E, F). Furthermore, EMT induced by ENO1 overexpression was significantly reversed after treatment with Ly294002 in both AGS and SGC7901 gastric cancer cells (Fig. 5C and D). These data suggest that ENO1 modulates GC cell proliferation and metastasis via regulation of AKT signaling pathway.
    Previous study (Fu et al., 2015) demonstrated that ENO1 regulates cell proliferation, migration and invasion through FAK-mediated PI3K/ AKT pathway in non-small cell lung cancer. Another study (Zhao et al., 2015) showed that knocking down ENO1 expression inhibites cancer cell phenotype via the p85 suppression-mediated inactivation of PI3K/ AKT pathway in endometrial carcinoma. A recent study (Dai et al., 2018) indicated that ENO1 regulates the malignant phenotype of pul-monary artery smooth muscle cells via the AMP-activated protein ki-nase (AMPK)/AKT pathway. In our present study, we showed that the effects of ENO1 on GC cell proliferation and metastasis are mediated by 
    AKT, but the exact mechanism underlying ENO1 activation of AKT in GC cells as we observed is not clear and warrants future investigation.
    Taken together, our present study not only indicates that ENO1 is upregulated in GC tissues, but also emphasizes its role in regulating GC cell proliferation and metastasis in vitro. Moreover, we revealed a po-tential mechanism for ENO1 in regulation of GC cell proliferation and metastasis through its participation in AKT signaling pathway. This may make it possible that ENO1 serves as a molecular therapeutic target for GC treatment.
    CRediT authorship contribution statement
    Liang Sun: Conceptualization, Writing - original draft. Ting Lu:
    Data curation. Kangjun Tian: Methodology. Diyuan Zhou: Formal
    analysis. Jingfeng Yuan: Data curation. Xuchao Wang: Methodology.
    Zheng Zhu: Formal analysis. Daiwei Wan: Methodology. Yizhou Yao:
    Formal analysis. Xinguo Zhu: Conceptualization, Funding acquisition, Writing - review & editing. Songbing He: Conceptualization, Funding acquisition, Writing - review & editing.
    Acknowledgements
    This work was supported by Project of Nature Science Foundation of China (81672348), National Science Foundation of Jiangsu Province of China (BK2016255), the Special Clinical Research Fund from Wu JiePing Medical Foundation (320.6750.17276), Six Major Talent Peak Project of Jiangsu Province of China (2015-WSW-014), Six One Project for Advanced Medical Talent of Jiangsu Province of China (LGY2016031), and Jiangsu Provincial Medical Youth Talent of China (QNRC2016735).
    Fig. 5. ENO1 enhances GC cell proliferation and metastasis through AKT signaling pathway. (A) Western blot analysis of p-AKT and AKT expression in cells stably transfected with control-shRNA (NC) or shRNA against ENO1 (KD). GAPDH as a loading control. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right). (B) Western blot analysis of p-AKT and AKT expression in cells stably transfected with empty vector (VEC) or plasmid encoding human ENO1 (ENO1). GAPDH as a loading control. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right). (C and D) Western blot analysis of indicated proteins in cells stably transfected with plasmid encoding human ENO1 in the presence or absence of Ly294002 (20 μM). GAPDH as a loading control. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right). (E) Colony formation assays were performed in SGC7901 cells stably transfected with plasmid encoding human ENO1 with or without Ly294002 (20 μM). Representative photographs are presented (left; magnification, x1) and the colonies were counted (right), presented as the mean ± S.E.M (n = 3). (F) Migration assays were performed in SGC7901 cells stably transfected with plasmid encoding human ENO1 with or without Ly294002 (20 μM). Representative photographs are presented (left; magnification, x200) and the number of migratory cells (right) were counted, presented as the mean ± S.E.M (n = 3). Statistical significance was determined by a two-tailed Student t-test. *P-values < 0.05, **P-values < 0.01.