4A). Consistent with the results of the in vitro Matrigel invasion assay, transfection with miR-205 precursor significantly inhibited the distance of OE21 cell migration, while transfection with anti-miR-205 inhibitor tended to promote in vitro wound done healing, though it was not significant (Figure (Figure4B4B). Figure 4 MiR-205 reduces epithelial-mesenchymal transition(EMT) through regulating zinc finger E-box binding homeobox2 (ZEB2) expression. Knockdown of miR-205 by transfection with 50 nM anti-miR-205 inhibitor significantly increased the invaded cell numbers on … miR-205 induces an epithelial-mesenchymal transition (EMT)-like phenotype through regulating zinc finger E-box binding homeobox 2 (ZEB2) expression Consistent with this, knockdown of miR-205 by anti-miR-205 inhibitor transfection enhanced cellular expression of ZEB2 but not ZEB1 in OE21 cells (Figure (Figure4C).

4C). On the other hand, overexpression of miR-205 by its precursor did not have impact on the expression of ZEBs. Downregulation of miR-205 decreased cellular E-cadherin expression, and instead, N-cadherin appeared in the OE21 cells transfected with anti-miR-205 inhibitor (Figure (Figure4C),4C), indicating acquisition of the EMT-like phenotype [16]. Overexpression of miR-205 by its precursor did not affect the expression levels of E- and N-cadherin. Again, transfection of anti-miR-205 inhibitor but not miR-205 precursor reduced cellular expression of phospho-Akt, consistent with recent studies [20,21].

miR-205 directly targets ZEB2 Co-transfection of the reporter plasmid along with miR-205 precursor resulted in a significantly reduced ZEB2-3′-UTR-luciferase expression, suggesting that miR-205 is likely to target ZEB2 directly (Figure (Figure5A).5A). In reporter assay using the ZEB1 3′-UTR, however, miR-205 precursor was unable to reduce the luciferase reporter expression (Figure (Figure5A5A). Figure 5 MiR-205 directly targets ZEB2 and miR-205 expression level in invasive ESCC tumors with poor differentiation is higher than in intraepithelial ESCC tumors. Activities of the firefly luciferase with the ZEB1 or ZEB2 3′-untranslated region (UTR) in the … miR-205 is not involved in cellular differentiation of ESCC tumors There were 7 intraepithelial and 21 invasive ESCC patients. The invasive ESCCs were composed of each 7 well, 5 moderate and 9 poor differentiation, respectively.

The miR-205 expression in ESCC tumor samples was assessed using real-time RT-PCR. There were no significant differences in the relative miR-205 expression levels between the AV-951 ESCC tumors and their paired surrounding non-tumor tissues, though miR-205 was highly expressed in the tumors of 16 of 28 cases examined (Figure (Figure5B).5B). The miR-205 expression did not differ significantly between intraepithelial and invasive ESCC samples.