a Schematic diagram of four potential Gli1 binding sites (BS1, BS2, BS3, and BS4) in the FoxM1 promoter

a Schematic diagram of four potential Gli1 binding sites (BS1, BS2, BS3, and BS4) in the FoxM1 promoter. by which Gli1 regulates FoxM1 in CRC. Methods Western blotting and immunohistochemistry (IHC) were used to evaluate FoxM1 and Gli1 protein expression, respectively, in CRC tissues and matched adjacent normal mucosa. BrdU (5-bromo-2-deoxyuridine) and clone formation assays were used to clarify the influence of FoxM1 on CRC cell growth and proliferation. Chromatin immunoprecipitation (ChIP) and luciferase experiments were performed to explore the potential mechanisms by which Gli1 regulates FoxM1. Additionally, the protein and mRNA expression levels of Gli1 and FoxM1 in six CRC cell lines were measured using Western blotting and real-time PCR. Finally, the effect of Hh signaling around the expression of FoxM1 was analyzed in cell biology experiments, and the effects of Hh signaling activation and FoxM1 inhibition around the distribution of CRC cells among cell cycle phases was assessed by circulation cytometry. Results Gli1 and FoxM1 were abnormally elevated in human CRC tissues compared with matched adjacent normal mucosa samples, and FoxM1 is usually a downstream target gene of the transcription factor Gli1 in CRC and promoted CRC cell growth and proliferation. Moreover, the aberrant activation of Hh signaling promoted CRC cell proliferation by directly binding to the promoter of FoxM1 and transactivating the activity of FoxM1 in CRC cells. Conclusion The dysregulation of the Hh-Gli1-FoxM1 axis is essential for the proliferation and growth of human CRC cells and offers a potent target for therapeutic intervention in CRC. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0491-7) contains supplementary material, which is available to authorized users. promoter As in our previous gene expression profile analyses (“type”:”entrez-geo”,”attrs”:”text”:”GSE54936″,”term_id”:”54936″GSE54936 and “type”:”entrez-geo”,”attrs”:”text”:”GSE53464″,”term_id”:”53464″GSE53464) [25, 26], FoxM1 was downregulated after the Hh-Gli signaling pathway was inhibited. In this study, we also found that FoxM1 promoted CRC cell proliferation. Thus, we hypothesized that FoxM1 is usually a target gene of the Hh-Gli1 signaling pathway in CRC. To determine whether Gli1 regulates FoxM1 expression by directly binding to the promoter of FoxM1, we recognized four potential Gli1 binding sites (Gli1 binding Cephalothin motif, 5-GACCACCCA-3) in the gene promoter of FoxM1 using MatInspector professional version 7.2 [36]. These putative Gli1 binding sites (BS1: ?1992?~??1980, BS2: ?1755?~??1743, BS3: ?1647?~??1635 and BS4: ?216?~??204) are located upstream of the transcriptional start site of the gene from ?1992?bp to ?204?bp (Fig.?2a). Among these four binding sites, BS1, BS2 and BS3 contained two nucleic acids that differed from the consensus sequence and shared a 78% homology with this consensus sequence, whereas BS4 exhibited only one differing Cephalothin base pair and shared an 89% homology with the consensus sequence. We performed ChIP studies in HT29 cells using Gli1 and Gli2, a homolog of Gli1, specific antibodies and an IgG control antibody. Although the Gli1 antibody immunoprecipitated the FoxM1 promoter containing the BS4 region, the Gli1 homolog Gli2 did not, which demonstrated that Gli1 directly bound to the FoxM1 promoter (Fig.?2b). To further confirm the role of Gli1 in the regulation of FoxM1 transcription, we generated five luciferase reporter vectors driven by the potential Gli1 binding site-containing FoxM1 promoter: Full-pFoxM1 (?2621?~?+1), Full-pFoxM1-BS4-Mut (?2621?~?+1-Mut), Frag-pFoxM1-BS4 (?2621?~??465), Frag-pFoxM1-BS4 (?512?~?+1) and Frag-pFoxM1-BS4-Mut (?512?~?+1-Mut) (Fig.?2c) and performed luciferase reporter assays using LoVo cells. As expected, the overexpression of Gli1 significantly increased the luciferase activity driven by the full-length (Full-pFoxM1) or the short BS4-containing FoxM1 promoter (Frag-pFoxM1-BS4), but not the Frag-pFoxM1CBS4 promoter, in which the Gli1 effective binding site region BS4 was deleted, or the BS4-mutated full-length FoxM1 (Full-pFoxM1-BS4-Mut) promoter (Fig.?2d). In addition, the mutated short BS4-containing promoter (Frag-pFoxM1-BS4-Mut) significantly decreased the luciferase activity compared with the Frag-pFoxM1-BS4 promoter (Fig.?2d). These results suggest that FoxM1 is a target gene of the Hh signaling EPOR pathway and that Gli1 transcriptionally activates FoxM1 by directly binding to the promoter of FoxM1 at BS4. Open in a separate window Fig. 2 Gli1 transactivates the FoxM1 promoter. a Cephalothin Schematic diagram of four potential Gli1 binding sites (BS1, BS2, BS3, and BS4) in the FoxM1 promoter. The 9-base pair sequence of the Gli1 binding site and the sequences of four Gli1 binding sites identified in the FxoM1 promoter are shown. b Cephalothin Chromatin was isolated from HT29 cells, and ChIP assays were performed with goat IgG control, Gli1-specific and Gli2-specific antibodies. DM, DNA marker. c Schematic diagram of a series of FoxM1-luciferase constructs. BS4-Mut, binding site 4 mutation; BS4, binding site 4 deletion. d FoxM1 luciferase constructs, as indicated, were transfected into LoVo cells together with full-length V5-Gli1 plasmid or control vector for 48?h and subjected to a luciferase reporter assay. The results were normalized to the Renilla.