Our preliminary investigations suggested that GLI1 plays a critical role in the EMT phenotype of HCC cells and the results of the current study strongly indicated that CtBP2 might be involved in EMT in HCC. Therefore, we next investigated whether the transcription factor GLI1 regulates CtBP2 expression. We stably transfected Huh7 cells with a GLI1 expressing vector or an empty control vector and observed that elevated GLI1 expression significantly enhanced CtBP2 expression (Figure 5A). Next, using MatInspector professional version 7.2, we identified several potential GLI binding sites in the CtBP2 promoter (Figure 5B). To determine whether GLI1 could directly interact with the CtBP2 promoter and to identify the core promoter region of CtBP2, we cloned four CtBP2 promoter fragments (pGL3−2000, −2000/−1 bp; pGL3−1307, −1307/−1 bp; pGL3−675, −675/−1 bp; pGL3−1350, −1350/−652 bp) into a luciferase reporter vector and conducted a luciferase reporter assay in Huh7 cells stably transfected with the GLI1 expressing plasmid. The four reporter plasmids each displayed different levels of promoter activity, but all displayed notably stronger promoter activity than the pGL3-basic control plasmid (Figure 5C). The pGL3−1307 plasmid
