26 Background: HOXB9 is a member of class I HOX genes and is known to be overexpressed in human breast cancer and related to EMT, tumor angiogenesis, and radio-resistance (PNAS 2010), being significant prognostic factor in breast cancer patients. (Ann Surg Oncol 2012). Methods: We analyzed the upstream promoter region to determine the critical sequence for HOXB9 transcription. The multiple lengths of HOXB9 promoter region were cloned into pGL3 luciferase vector and each of them were checked by luciferase assay. Then made E2F1 overexpressed cell-lines: MCF7, MCF10A and MDA-MB231 to check HOXB9 gene expression. ChIP assay performed using E2F1 induced MCF7 cell-line model and EMSA made with T47D cells and E2F1 antibody. Results: We demonstrated luciferase activity of MDA-MB231 cells and overexpressed HOXB9 was determined at the region from -404 to -392 bps, showing that the mentioned region plays the critical role for the transcription of HOXB9. Computer prediction system for the binding ability to critical sequence raised several candidate: E2F1, PAX-5, P53, ETS-1, NFY-A, Sp-1, which are known to be related with cancer progression and we confirmed these genes could induce HOXB9. E2F1 overexpressed breast cancer cell-lines indicate high expression of HOXB9 show the correlation between E2F1 and HOXB9 expression. Luciferase assay method done by constructing pGL3 with HOXB9 critical sequence and analyzed using wild-type MCF7 and E2F1 induced MCF7 cells showed high luciferase activity of HOXB9 critical sequence plasmid in E2F1 induced MCF7 cell-line by compare to WT. ChIP assay and EMSA assay methods performed using E2F1 overexpressed MCF7 and T47D cell-lines model revealed high precipitation rate with E2F1 antibody and HOXB9 expression than negative control. Conclusions: HOXB9 is plays the role as the accelerator of breast cancer progression and discovering the properties of the gene and related factors will help to understand new signaling pathways of breast cancer development. E2F1 is a strong candidate direct binds to HOXB9 critical sequence and correlates with HOXB9 expression. Manipulating of E2F1 expression may regulate HOXB9 and reduce cancer progression and metastasis.
MicroRNA-126-5p Inhibits the Migration of Breast Cancer Cells by Directly Targeting CNOT7
