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  • br N C Representative images

    2020-07-08


    (N). (C) Representative images of ENO1 IHC staining in GC tumors and surrounding normal tissues (top, magnification, x200; bottom, magnification, x400). (D) Scatter plot analysis of ENO1 IHC scores in 76 gastric tumor tissues and adjacent normal tissues. (E) Scatter plot analysis of ENO1 IHC scores in GC tissues with or without LNM. Data are represented as mean ± S.E.M. Statistical significance was determined by a two-tailed Student t-test. **P-values < 0.01, ***P-values < 0.001.
    treatment with Ly294002 in both AGS and SGC7901 gastric cancer cells. Similarly, colony formation assays and migration assays showed that the enhanced proliferation and migration ability induced by overexpression of ENO1 was obviously impaired after incubation with Ly294002 in SGC7901 GC AR-13324 (Fig. 5E, F). These data suggest that ENO1 modulates GC cell proliferation and metastasis via regulation of AKT signaling pathway.
    4. Discussion
    It is widely accepted that increased aerobic glycolysis is considered as a hallmark of cancer and cancer cells acquire characteristic changes in glucose metabolism to support their unrestricted proliferation and metastasis. So metabolic manipulation, such as targeting key glycolytic enzymes, will be a promising therapeutic approach in cancer treatment (Hanahan and Weinberg, 2011; Boroughs and DeBerardinis, 2015). The ENO1, as a prominent enzyme of the glycolytic pathway, catalyzes the 
    dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, which generates ATP to support cancer cell proliferation and metastasis (Vander Heiden et al., 2009). Recently, accumulating evidences showed that ENO1 expressed aberrantly in several types of cancers, such as pancreatic cancer, glioma, head and neck cancer, breast cancer and colorectal cancer (Cappello et al., 2009; Song et al., 2014; Tsai et al., 2010; Shih et al., 2010; Zhan et al., 2017; Zhao et al., 2015).
    Studies focusing on ENO1 in GC are scanty. Previous studies (Tsai et al., 2010; Shih et al., 2010) have provided evidence that ENO1 was upregulated in gastric tumor tissues and its overexpression promoted GC cell proliferation and drug resistance to cisplatin (Qian et al., 2017; Qiao et al., 2018; Liu et al., 2015), indicating that ectopic ENO1 ex-pression contributes to the development and progression of GC. How-ever, the concrete molecular mechanisms of ENO1 in GC cell pro-liferation and metastasis remains unclear and need to be explored.
    In the present study, we provided further evidence that the ex-pression of ENO1 in GC tissues was higher than that in the adjacent
    Fig. 2. ENO1 knockdown and over-expression efficiency in human GC cells. (A) The protein levels of ENO1 in GC cells stably transfected with control-shRNA (NC) or shRNA against ENO1 (KD) were tested by Western blot. The bands were quantified and presented as the mean ± S.E.M of three independent experiments (right). (B) The protein levels of ENO1 in GC cells stably transfected with empty vector (VEC) or plasmid encoding human ENO1 (ENO1) were tested by Western blot. The bands were quantified and presented as the mean ± S.E.M of three independent experi-ments (right). Statistical significance was de-termined by a two-tailed Student t-test. **P-values < 0.01.
    normal tissues. Moreover, clinical data analyses showed that the en-hanced ENO1 expression was significantly associated with Lauren type, LNM and TNM stage in GC patients, suggesting a possible involvement of ENO1 in GC initiation and progression. We also evidenced that si-lencing ENO1 weakened the ability of GC cell proliferation and me-tastasis while ENO1 overexpression did the opposite.
    Emerging literature suggests that EMT is involved in cell migration and invasion (Gonzalez and Medici, 2014). The loss of epithelial-asso-ciated markers such as E-cadherin and overexpression of mesenchymal- 
    Fig. 3. Effects of ENO1 on GC cell proliferation and metastasis in vitro. (A) Colony formation assays were performed in AGS cells stably transfected with control-shRNA (NC) or shRNA against ENO1 (KD). Representative photographs are presented (left; magnification, x1) and the colonies were counted (right), presented as the mean ± S.E.M (n = 3). (B) Colony formation assays were conducted in SGC7901 cells stably transfected with empty vector (VEC) or plasmid encoding human ENO1 (ENO1). Representative photographs are presented (left; magnification, x1) and the colonies were counted (right), presented as the mean ± S.E.M (n = 3). (C) Migration assays were conducted in AGS NC or KD cells. 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). (D) Migration assays were conducted in SGC7901 VEC or ENO1 cells. 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 < 0.01.