miR-204 inhibits invasion and epithelial-mesenchymal transition by targeting FOXM1 in esophageal cancer

Abstract

MicroRNAs (miRNAs), endogenous noncoding small RNAs, have been reported to play crucial roles in epithelial-mesenchymal transition (EMT) in cancers. Deregulation of microRNA-204 (miR-204) has been documented in many cancers, but its role in the development of esophageal cancer (EC) has not been studied. Here, we reported the role of miR-204 in invasion and EMT in EC. We identified an inverse correlation between miR-204 expression level and the invasion and EMT phenotype of EC cells, and up-regulation of miR-204 inhibited invasion and EMT phenotype of EC cells. Furthermore, we showed that forkhead box protein M1 (FOXM1) was a direct target gene of miR-204, and miR-204 regulated invasion and EMT in EC by acting directly on the 3'UTR of FOXM1 mRNA and suppressing its protein expression. We also explored the anti-tumor effect of miR-204, and found that overexpression of miR-204 suppressed the growth of esophageal tumors in vivo. These findings suggest that miR-204 might be a suppressor of invasion and EMT in EC, which offers a novel potential therapeutic target for EC.

Keywords: Esophageal cancer; epithelial-mesenchymal transition; forkhead box protein M1 (FOXM1); invasion; miR-204.

Figure 1

The expression level of miR-204 in primary EC cases and cell lines. A. qRT-PCR shows that miR-204 was frequently up-regulated in 21 primary ES tissues compared with their adjacent normal tissues. B. Up-regulated miR-204 was detected in EC109 and TE10 cell line compared with human esophageal epithelial cells (HEEpiC). **P < 0.01.

Figure 2

miR-204 inhibited invasive and EMT of EC cell lines. A and B. EC109 and TE10 cells transfected with miR-204 mimics had a significantly lower invasion capacity than the control group. C and D. Compared with the control group, EC109 and TE10 cells transfected with miR-204 mimic had dramatic decreases in N-cadherin, fibronectin and vimentin expression and significant increases in E-cadherin and α1-catenin expression. **P < 0.01.

Figure 3

FOXM1 is a direct target of miR-204. A. Bioinformatics-based target prediction analysis showed that FOXM1 is a potential target gene of miR-204 and the binding site is on the 3’UTR of FOXM1 mRNA. B. Luciferase reporter assay showed that in the miR-204 group, the luciferase activity driven by 3’UTR of FOXM1 mRNA is significantly lower than that in the miR-NC or 3’UTR-MUT group. C. A negative correlation was observed between miR-140 and FOXM1 mRNA in clinical samples (r = -0.861; P < 0.0001). D. The miR-204 group had a significantly low expression of FOXM1 protein compared with the miR-NC group. Relatively, the anti-miR-204 group had an obvious high expression of FOXM1 protein compared with the anti-miR-NC group. E. The transwell invasion assay showed that in the Si-FOXM1 group, the number of invaded cells decreased significantly compared to cells transfected with scramble siRNA (Si-control). F. The Si-FOXM1 group had dramatic decreases in N-cadherin, fibronectin and vimentin expression and significant increases in E-cadherin and α1-catenin expression compared to the Si-control group. **P < 0.01, ***P < 0.001.

Figure 4

miR-204 suppressed tumorigenesis of EC in vivo. A. In the miR-204 group, the tumor volume was significantly decreased than that in the control group. B. After 4 weeks, the tumor weight was significantly decreased in the miR-204 group than that in the control group. C. miR-204 inhibited the expression of FOXM1. D. miR-204 decreased N-cadherin, fibronectin and vimentin expression and increased N-cadherin, fibronectin and vimentin expression compared to the control group. *P < 0.05, **P < 0.01.