Research Paper Volume 13, Issue 12 pp 16043—16061

HMGA1 promotes gastric cancer growth and metastasis by transactivating SUZ12 and CCDC43 expression

HMGA1 directly binds to the SUZ12 or CCDC43 promoter and up-regulated the SUZ12 or CCDC43 promoter activity. (A) The vector and HMGA1 plasmid were transfected into AGS and BGC-823 cells. The HOXD9, E-cadherin, FOXK1, SUZ12, CCDC43 and HMGA1 expression levels were detected in AGS and BGC-823 cell lines by western blot assay. (B, C) The relationship between HMGA1 and SUZ12 or CCDC43 levels was analyzed by GEPIA bioinformatics tool. (D) List of consensus HMGA1 sequence. (E, F) Binding of HMGA1 to the SUZ12 or CCDC43 promoter in vivo. ChIP assays were done with AGS and BGC-823 cells treated with anti-HMGA1 or IgG. (G, H) HMGA1 transactivates SUZ12 or CCDC43 promoter activities in AGS cells. The SUZ12 or CCDC43 promoter construct was cotransfected with HMGA1 or vector, and the relative luciferase activity was determined. **, P I, J) Selective mutation analyses identified HMGA1-responsive regions in the SUZ12 or CCDC43 promoter. Mutated SUZ12 or CCDC43 promoter constructs were cotransfected with HMGA1 and relative luciferase activities were determined. *, P > 0.05; **, P K) Western blot assay were used to detect the expression of SUZ12, CCDC43 and HMGA1. (L) The protein levels of SUZ12 and CCDC43 in AGS and BGC-823 cells with three treatments [Scrambled (Scr) siRNA, HMGA1 siRNA 1 and HMGA1 siRNA 2] determined by western blot analysis.

Figure 2. HMGA1 directly binds to the SUZ12 or CCDC43 promoter and up-regulated the SUZ12 or CCDC43 promoter activity. (A) The vector and HMGA1 plasmid were transfected into AGS and BGC-823 cells. The HOXD9, E-cadherin, FOXK1, SUZ12, CCDC43 and HMGA1 expression levels were detected in AGS and BGC-823 cell lines by western blot assay. (B, C) The relationship between HMGA1 and SUZ12 or CCDC43 levels was analyzed by GEPIA bioinformatics tool. (D) List of consensus HMGA1 sequence. (E, F) Binding of HMGA1 to the SUZ12 or CCDC43 promoter in vivo. ChIP assays were done with AGS and BGC-823 cells treated with anti-HMGA1 or IgG. (G, H) HMGA1 transactivates SUZ12 or CCDC43 promoter activities in AGS cells. The SUZ12 or CCDC43 promoter construct was cotransfected with HMGA1 or vector, and the relative luciferase activity was determined. **, P < 0.05; ***, P < 0.01. (I, J) Selective mutation analyses identified HMGA1-responsive regions in the SUZ12 or CCDC43 promoter. Mutated SUZ12 or CCDC43 promoter constructs were cotransfected with HMGA1 and relative luciferase activities were determined. *, P > 0.05; **, P < 0.05; ***, P < 0.01. (K) Western blot assay were used to detect the expression of SUZ12, CCDC43 and HMGA1. (L) The protein levels of SUZ12 and CCDC43 in AGS and BGC-823 cells with three treatments [Scrambled (Scr) siRNA, HMGA1 siRNA 1 and HMGA1 siRNA 2] determined by western blot analysis.