Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer

Figure 1 Ubiquitin-like modifier activating enzyme 2 expression in gastric cancer tissues and normal gastric tissues. A: Western blotting showed that Uba2 expression was higher in gastric cancer tissues than in paired normal gastric tissues; B: Weak nuclear Uba2 staining in normal gastric mucosa; C: Low nuclear Uba2 expression in adenocarcinoma; D: High nuclear Uba2 expression in signet ring cell carcinoma (magnification: 200 ×). N: Normal gastric tissues; T: Gastric cancer tissues; Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 2 Expression of ubiquitin-like modifier activating enzyme 2 in different cell lines analyzed by Western blotting. A: Uba2 expression in SGC-7901, BGC-823 and MGC80-3 GC cell lines and the GES-1 normal gastric epithelial cell line; B: Knock-down efficiency of Uba2 in BGC-823 cells; C: Over-expression of Uba2 in SGC-7901 cell line. ^aP < 0.05 vs Control; ^bP < 0.01 vs EV. EV: Empty vector; Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 3 Ubiquitin-like modifier activating enzyme 2 promotes cell growth and colony formation in gastric cancer. A: MTT assay showed that knock-down of Uba2 (siUba2) inhibited growth of BGC-823 cells, and over-expression of Uba2 promoted growth of SGC-7901 cells; B: Colony formation assay showed down-regulation of colony number in BGC-823 siUba2 cells and up-regulation in SGC-7901 cells with Uba2 over-expression. Data are shown as mean ± SD from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs Control; ^dP < 0.05, ^eP < 0.01 vs EV. EV: Empty vector; Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 4 Ubiquitin-like modifier activating enzyme 2 facilitates cell cycle progression and regulates expression of cell cycle-related proteins. A: Cell cycle analysis by flow cytometry in BGC-823 and SGC-7901 cells. ^aP < 0.05 vs Control; ^bP < 0.05 vs EV; B: Expression of cyclinD1, P21, P27, CDK4, and Bcl-2. All experiments were performed in triplicate. CDK4: Cyclin-dependent kinase 4; EV: Empty vector; Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 5 Ubiquitin-like modifier activating enzyme 2 promotes gastric cancer migration and invasion in vitro. A: Wound healing assay showed that knock-down of Uba2 inhibited migration of BGC-823 cells, while over-expression of Uba2 promoted migration of SGC-7901 cells (magnification: 50 ×). Data are shown as mean ± SD from three independent experiments; B: Transwell assays showed that knock-down of Uba2 inhibited migratory and invasion properties in BGC-823 cells, and over-expression of Uba2 in SGC-7901 cells showed the opposite effects (magnification: 100 ×). Data are shown as mean ± SD from three independent experiments; C: Alterations of MMP2, c-Myc, and MMP7 in BGC-823 and SGC-7901 cells. ^aP < 0.05, ^bP < 0.001 vs Control; and ^cP < 0.05, ^dP < 0.001 vs EV. EV: Empty vector; MMP: Matrix metalloproteinase; Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 6 Impact of ubiquitin-like modifier activating enzyme 2 expression on the expression of E-cadherin and vimentin. Western blotting showed that expression of E-cadherin was up-regulated and vimentin was decreased in BGC-823 cells transfected with siRNA, while expression of E-cadherin was down-regulated and vimentin was up-regulated in SGC-7901 cells with Uba2 over-expression. Uba2: Ubiquitin-like modifier activating enzyme 2.

Figure 7 Alterations in Wnt/β-catenin signaling and related protein expression levels in BGC-823 and SGC-7901 cells. A: Western blotting showed that expression of proteins related to the Wnt/β-catenin signaling pathway was altered in BGC-823 cells transfected with siRNA and SGC-7901 cells with Uba2 over-expression; B: Luciferase reporter assays of BGC-823 cells transfected with siRNA and SGC-7901 cells with Uba2 over-expression after transfection with TOP Flash or FOP Flash and Renilla pRL-TK plasmids. Reporter activity was normalized to Renilla luciferase activity. ^aP < 0.01 vs Control; ^bP < 0.01 vs EV. EV: Empty vector; Uba2: Ubiquitin-like modifier activating enzyme 2.