Down-regulation of TWIST decreases migration and invasion of laryngeal carcinoma Hep-2 cells by regulating the E-cadherin, N-cadherin expression

Abstract

Purpose: The transcription factor TWIST is an important factor in regulation of the epithelial-mesenchymal transition (EMT), which represents the primary stages during the metastasis of tumors. To identify the role of TWIST in the regulation of metastasis in laryngeal carcinoma Hep-2 cells, we investigated whether the alteration of TWIST has an effect on the Hep-2 cells morphology and whether the alteration of TWIST has an effect on the expression of E-cadherin, N-cadherin as well as the ability of cell motion, migration, and invasion.

Methods: Morphological changes of Hep-2 cells that were transfected a mircoRNA against TWIST vector were observed by the reserved microscope. Reverse transcription-polymerase chain reaction was performed in order to examine the mRNA expression of TWIST, E-cadherin, and N-cadherin. Western blotting was performed to examine the protein expression of TWIST, E-cadherin, and N-cadherin. Cell motion ability was examined by Scratch-wound assay. Transwell(™) chamber assays were used to determine cell migration and invasion.

Results: Transfecting a mircoRNA down-regulated TWIST expression at mRNA and protein levels. Down-regulation of TWIST expression induced morphological changes, such as the inversion of the EMT. Moreover, down-regulation of TWIST expression up-regulated E-cadherin and down-regulated N-cadherin expressions at mRNA and protein levels, respectively. Furthermore, we confirmed that down-regulation of TWIST expression decreased the motion, invasion, and migration ability of the Hep-2 cells.

Conclusions: Down-regulation of TWIST expression decreases migration and invasion of laryngeal carcinoma Hep-2 cells by regulation of the E-cadherin, N-cadherin expression.

Fig. 1

Down-expression of TWIST changed morphology of Hep-2 cells. Morphology of cells in different groups was observed by reserved microscopy (×200 magnification). Morphologic changes from scattered and fibroblast-like growth in control group to tightly packed colonies in miRNA-TWIST vector group. The phenotype of Hep-2 cell changed from the mesenchymal cell to epithelial cells

Fig. 2

The alteration in the expression of TWIST, E-cadherin, and N-cadherin at mRNA level in control group, miRNA-TWIST(−)vector group, and miRNA-TWIST(+)vector group, respectively, was assessed by reverse transcription-polymerase chain reaction analysis

Fig. 3

The alteration in expression levels of TWIST, E-cadherin, and N-cadherin protein in different groups by western blotting analysis. aThe alteration in expression of TWIST, E-cadherin, and N-cadherin at protein level in control group, miRNA-TWIST(−)vector group, and miRNA-TWIST(+)vector group, respectively. b–d The optical density value of TWIST (b), E-cadherin (c), and N-cadherin (d) each lane using National Institutes of Health software Image J system was demonstrated in different groups. Each data point in the figure represents the mean ± standard deviation of 3 separate experiments. All experiments were conducted 3 times

Fig. 4

Down-expression of TWIST inhibited cells motion ability in Hep-2 cells at different time points (0, 6, 12, 24 h) was examined by scratch-wound assay (×40 magnification)

Fig. 5

Down-expression of TWIST inhibited cell migration and invasion in Hep-2 cells in vitro migration and invasion assays. Migration (upper) and invasion (bottom) were examined by the transwell chambers assay in control group, miRNA-TWIST(−)vector group, miRNA-TWIST(+)vector group, respectively (×100 magnification). Each datum point in the figure represented the mean ± standard deviation of 3 separate experiments. All experiments were conducted 3 times