Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial-mesenchymal transition regulator Twist1

Abstract

E-cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial-mesenchymal transition (EMT) mechanism-mediated invasion and metastasis, Twist1 plays an important role through its regulation of E-cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E-cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down-regulate E-cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.

Fig 1

Positive Twist1 expression was detected in HCC cytoplasm and nucleus by immunostaining, and Twist2 expression was only detected in the cytoplasm. Nuclei expression for Twist2 was not detected. Twist1 was expressed in both the cytoplasm and nuclei, but Twist2 was only detected in the cytoplasm.

Fig 2

Twist1 and Twist2 expression level in HCC cell lines. RT-PCR was used to screen the level of Twist1 and Twist2 expression in the HCC cell lines HepG2, PLC, SMMC7221 and Bel7402. HepG2 and PLC had a low level of Twist1 expression and a low level of Twist2 expression. Bel7402 presented a high level of Twist1 expression, while SMMC7221 presented a high level of Twist2 expression.

Fig 3

Effect of ectopic expression of Twist1 and Twist2 by pcDNA3.1 in the HCC cell lines HepG2 and PLC cells. (A) We determined that the expression of the epithelial marker E-cadherin was down-regulated after Twist1 transfectant, which was detected by Western blot analysis. For the Twist2 transfectant, E-cadherin showed no significant differences between the transfect and control groups for HepG2 and PLC cells. (B) Based on zymographic assays, the activities of MMP2 and MMP9 in HepG2 and PLC cells were significantly higher in the Twist1 up-regulation group than with the control group, but Twist2 did not present the same changes after transfection. (C) To investigate further the effects of Twist1 and Twist2 activation on cell invasion, we used transwell invasion assay to analyse the different influences between Twist1 and Twist2. We studied the invasion ability of HepG2 and PLC cells after performing Twist1 and Twist2 ectopic transfection. The quantitative analysis suggests a significant difference between the Twist1 transfection groups and the control group (P < 0.05). Twist2, however, showed no significant result in HepG2 and PLC cells (P > 0.05).

Fig 4

E-cadherin, MMP2 and MMP9 expression in HCC. Brown or yellow staining was observed in the membrane and cytoplasm. E-cadherin was located in membranes. MMP2 and MMP9 were located in the cytoplasm.

Fig 5

Correlation analysis between Twist1(cytoplasm), Twist1(nuclear), Twist2, E-cadherin, MMP2 and MMP9 expression was tested using the Pearson correlation test. Results show that the Twist1 (nuclear) is correlated with Twist1(cytoplasm), E-cadherin and MMP9 (*P < 0.05).

Fig 6

Kaplan–Meier survival analysis in HCCs. The P-value of Twist1 (cytoplasm) was 0.000, Twist1 (nuclear) was 0.002, Twist2 was 0.336, E-cadherin was 0.002, MMP2 was 0.805 and MMP9 was 0.039.