Figure 6

HoxD10 transcriptionally regulates the expression of IGFBP3 in gastric cancer cells. (A). Schematic representation of IGFBP3 putative promoter with HoxD10 binding sites (BS). Upstream 2.3-kb sequence from the transcription start site of IGFBP3 were input to PROMO software to predict the possible HoxD10 binding sites (H1-H5). IGFBP3 DNA fragments containing of HoxD10 BS (−2,251 to −2,073 for BS1, and −1,727 to −943 for BS2) were cloned into pGL3-promoter vector and generated luciferase reporter gene. (B). Chromatin immunoprecipitation assays. The chromatin was immunoprecipitated with normal rabbit IgG and HoxD10 antibody in BGC823 cells. Quantitative real-time PCR was performed to detect DNA fragments in the promoter regions of IGFBP3 (A1 for H1 and H2, A2 for H3, A3 for H4, A4 for H5 respectively). Results were expressed as fold change compared to rabbit IgG normalized to 1. The experiments were performed in triplicate. (C). Luciferase reporter gene assay. Gastric cancer cell lines (BGC823 and SGC7901) were transfected with pcDNA3.1-HoxD10 or empty vector pcDNA3.1, pGL3-promoter vector or pGL3-BS, and pRL-TK vector. The relative fold of firefly activity was expressed normalized to renilla activity in pGL3-promoter vector. The experiments were performed in triplicate. **P < 0.01 indicates of HoxD10 versus empty vector. B: □, Rabbit IgG; ■, HoxD10 antibody; C: □, BGC-vector; , BGC-HoxD10; , SGC-vector; ■, SGC-HoxD10.