ECM1 promotes migration and invasion of hepatocellular carcinoma by inducing epithelial-mesenchymal transition

Abstract

Background: Extracellular matrix protein 1 (ECM1) is a glycoprotein involved in many biologic processes. To determine the expression of ECM1 in hepatocellular carcinoma (HCC), and to study the role of ECM1 in inducing epithelia-mesenchymal transition (EMT) to analyze the effect of ECM1 on the migration and invasion of HCC cells.

Methods: The expression of ECM1 in HCC specimens was examined by immunohistochemistry staining, and the correlations were analyzed between the expression of ECM1 and the clinicopathological data. The ECM1 expression plasmid was transfected into Bel-7402 cells to induce exogenous overexpression of ECM1 protein. The changes of the expression of ECM1, EMT-related protein (E-cadherin, Vimentin), in Bel-7402 cells were detected by Western blot after transfection of ECM1; the wound healing and invasion assay in vitro were used to determine the role of ECM1 gene transfection on the ability of migration and invasive potential of Bel-7402 cells.

Results: Immumohistochemistry staining method displayed the ECM1 expression was positively associated with vascular invasion, TNM stage, and poor prognosis. A significant positive correlation was found between the expressions of ECM1 and Vimentin. After ECM1 overexpression, Western blot exhibited that the expression of E-cadherin was down-regulated and Vimentin expression was up-regulated in Bel-7402 cells; the wound healing and invasion assay showed that the migration and invasion potentials of Bel-7402 cells were significantly enhanced.

Conclusions: ECM1, which displayed a high expression in HCC specimens, was closely associated with clinicopathologic data and may promote migration and invasion of HCC cells by inducing EMT.

Keywords: Epithelia-mesenchymal transition; Extracellular matrix protein 1; Hepatocellular carcinoma; Invasion; Migration.

Fig. 1

a Positive immunostainings for ECM1 in HCC tissues. b Positive immunostainings for Vimentin in HCC tissues. Both ECM1 and Vimentin mainly expressed in the cytoplasm of HCC cells. Serial sections of tumor samples were made into 3-μm sections. Original magnification at ×200

Fig. 2

a Kaplan-Meier analysis of overall survival curve of HCC patients according to ECM1 expression. The HCC patients with ECM1-positive showed significantly shorter overall survival survival rates than those with ECM1-negative. b Kaplan-Meier analysis of disease-free survival curve of HCC patients according to ECM1 expression. The expression of ECM1 is associated with decreased disease-free survival rate

Fig. 3

a Representative pictures of ECM1 staining in Bel-7402 cells, ECM1 mainly expressed in the cytoplasm of cells. b Representative pictures of ECM1 staining in ECM1-Bel-7402 cells. The ECM1 level of Bel-7402 cells was significantly increased detected by immunohistochemical staining. Original magnification at ×200

Fig. 4

a Changes of ECM1 and EMT-related protein (E-cadherin and Vimentin) expression in Bel-7402 cells were detected by Western blot after ECM1 transfection. The increased expression of ECM1 could lead to the up-regulated expression of Vimentin protein and down-regulated expression of E-cadherin protein in Bel-7402 cells. b The relative quantity of three protein expressed levels in cells with significant difference (ECM1: t = 12.909, P < 0.01; Vimentin; t = 10.564, P < 0.01; E-cadherin: t = −8.558, P = 0.002). Relative quantity of three protein expressed levels in cells. Data are mean ± SD of three replicates. (P < 0.05)

Fig. 5

a Effect of ECM1 on the migration capacity of Bel-7402 cell by the wound healing assay at 24, 48, and 72 h (×40). b Statistics of analysis of migration rate date in Bel-7402 and ECM1-Bel-7402 cells. The wound healing assay revealed that the migration rates of the cells in the Bel-7402 group infected with pEGFP-N2-ECM1 were significantly increased compared with those without transfected group (F = 110.592, P < 0.01). Data are mean ± SD of three replicates. (P < 0.05)

Fig. 6

a, b Effect of ECM1 on the invasion capacity of Bel-7402 and ECM1-Bel-7402 cells by invasion assay (×200). c Statistics of analysis of cells per field in Bel-7402 and ECM1-Bel-7402 cells at 48 h. Compared with the control group, after the up-regulated expression of ECM1, the number of invading Bel-7402 cells was significantly increased (89.3 ± 2.5 cells/well vs 28.7 ± 2.5 cells/well) (t = 29.524, P < 0.01). Data are mean ± SD of three replicates (P < 0.05)