Paired-related homeobox 1 induces epithelial-mesenchymal transition in oesophageal squamous cancer

Abstract

Background: It is unclear that paired-related homeobox 1 (PRRX1) induces epithelial-mesenchymal transition (EMT) in oesophageal cancer and the specific function of PRRX1 in oesophageal cancer metastasis.

Aim: To assess the significance of PRRX1 expression and investigate the mechanism of EMT in oesophageal cancer metastasis.

Methods: Detect the expression of PRRX1 by immunohistochemistry in oesophageal tumour tissues and adjacent normal oesophageal tissues; the PRRX1 short hairpin RNA (shRNA) or blank vector lentiviral gene delivery system was transfected into cells; cell proliferation assay, soft agar colony formation assays, cell invasion and migration assays and animal studies were used to observe cells biological characteristics In vitro and in vivo; XAV939 and LiCl were used to alter the activity of Wnt/β-catenin pathway. Immunofluorescence staining and western blot analysis were used to detect protein expression of EMT markers and Wnt/β-catenin pathway.

Results: PRRX1 is expressed at high levels in oesophageal cancer specimens and is closely related to tumour metastasis in patients with oesophageal cancer. Regulation of PRRX1 expression might exert obvious effects on cell proliferation, especially the migration and invasion of oesophageal cancer cells. Moreover, silencing PRRX1 expression using a shRNA produced the opposite effects. In addition, when PRRX1 was overexpressed, inhibition of the Wnt/β-catenin pathway with XAV939 negated the effect of PRRX1 on EMT, whereas when PRRX1 was downregulated, activation of the Wnt/β-catenin pathway with LiCl impaired the effect on EMT.

Conclusion: PRRX1 is upregulated in oesophageal cancer is closely correlated with cancer metastasis. Additionally, PRRX1 induces EMT in oesophageal cancer metastasis through activation of Wnt/β-catenin signalling.

Keywords: Cancer metastasis; Epithelial-mesenchymal transition; Oesophageal squamous cancer; Paired-related homeobox 1.

Figure 1

Paired-related homeobox 1 expression is upregulated in oesophageal cancer and related to shorter overall survival. A: Immunohistochemical detection of the expression of paired-related homeobox 1 (PRRX1) in oesophageal cancer specimens with a high (× 200); B: Middle (× 200); C: Low grade (× 200); D: Immunohistochemical detection of the expression of PRRX1 in adjacent normal oesophageal tissues (× 200); E: Logrank test and Kaplan-Meier analysis of the association of PRRX1 expression with the overall survival of patients with oesophageal cancer. ^aP < 0.05. PRRX1: Paired-related homeobox 1.

Figure 2

Paired-related homeobox 1 not only induces epithelial-mesenchymal transition but also regulates Wnt/β-catenin signalling. A and B: The protein levels of paired-related homeobox 1, epithelial-mesenchymal transition markers (E-cadherin, Vimentin, and N-cadherin), total β-catenin and nuclear β-catenin in various groups of EC9706 and EC109 cells were examined using western blotting. ^aP < 0.05. PRRX1: Paired-related homeobox 1.

Figure 3

Paired-related homeobox 1 modulates proliferation, colony formation, migration and invasion in oesophageal cancer cells. A: The MTT assay. Paired-related homeobox 1 (PRRX1) affected EC9706 cell proliferation; B: Soft agar colony formation assay. PRRX1 affected the ability of EC9706 cells to form colonies in soft agar, and the number of colonies was different in various groups of EC9706 cells; C: Cellular migration assays. PRRX1 affected the migration of EC9706 cells. The number of migrating EC9706 cells in various groups was different. Cells were stained with hematoxylin and eosin; D: Cellular invasion assays. PRRX1 affected the invasive properties of EC9706 cells. The number of invasive EC9706 cells in various groups was different. Cells were stained with hematoxylin and eosin. ^aP < 0.05. PRRX1: Paired-related homeobox 1.

Figure 4

Cellular immunofluorescence staining. The protein levels of epithelial-mesenchymal transition markers (E-cadherin, N-cadherin, and Vmentin) and β-catenin in cells were stained red, and the nucleus was stained blue with 4’,6-diamidino-2-phenylindole. PRRX1: Paired-related homeobox 1; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 5

Paired-related homeobox 1 regulates epithelial-mesenchymal transition via the WNT/β-catenin pathway. A: The protein levels of paired-related homeobox 1 (PRRX1), epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin, and N-cadherin), total β-catenin and nuclear β-catenin in different groups of EC109 and EC9706 cells (treated with 10 μM XAV939) were examined using western blotting; B: The protein levels of the EMT markers total β-catenin nuclear β-catenin, and PRRX1 in different groups of EC109 PRRX1(i) and EC9706 PRRX1(i) cells (treated with 20 mmol/L LiCl) were examined using western blotting. ^aP < 0.05. PRRX1: Paired-related homeobox 1.

Figure 6

Paired-related homeobox 1 downregulation inhibits cell proliferation and lung metastasis in vivo. A: Paired-related homeobox 1 (PRRX1) affected EC9706 cell proliferation in vivo. The tumour volume was measured weekly; B: The number of metastatic lung nodules was different in the various EC9706 groups. ^aP < 0.05. PRRX1: Paired-related homeobox 1.