Dbx2 exhibits a tumor-promoting function in hepatocellular carcinoma cell lines via regulating Shh-Gli1 signaling

Figure 1 Developing brain homeobox 2 expression is frequently up-regulated in tumor tissues than in non-tumor tissues from patients with hepatocellular carcinoma. A: Positive developing brain homeobox 2 (Dbx2) expression in tumor tissues and negative Dbx2 expression in non-tumor tissues as revealed by immunohistochemistry (×400); B and C: Dbx2 expression was significantly upregulated in tumor tissues compared with their matched adjacent non-tumor tissues as revealed by immunohistochemistry; D and E: Relative Dbx2 expression in tumor tissues to paired non-tumor tissues; F: There was more Dbx2 expression in hepatocellular carcinoma (HCC) cells than in normal hepatic epithelial LO2 cells; G and H: Dbx2 expression was significantly related with gender and tumor size.

Figure 2 Developing brain homeobox 2 promotes cell growth in vitro and in vivo. A-C: Ectopic expression of developing brain homeobox 2 (Dbx2) in SMMC-7721 and Huh7 cell lines promoted cell growth as indicated by a cell viability assay and colony formation assay. The results were reversed in HepG2 and Huh7 cell lines with knockdown of Dbx2; D: Ectopic Dbx2 expression in SMMC-7721 cells promoted tumor growth and knockdown of Dbx2 in HepG2 cells inhibited tumor growth in NOD/SCID mice. ^aP < 0.05; ^bP < 0.01.

Figure 3 Developing brain homeobox 2 regulates cell cycle and inhibits cell apoptosis in hepatocellular carcinoma cells. A and B: Ectopic expression of developing brain homeobox 2 (Dbx2) induced cell cycle into S phase; C: Ectopic expression of Dbx2 regulated cell cycle protein expression; D and E: Ectopic expression of Dbx2 inhibited cell apoptosis; F: Ectopic expression of Dbx2 downregulated apoptotic protein expression. The results were reversed after Dbx2 knockdown.

Figure 4 Developing brain homeobox 2 promotes cell migration and invasion in hepatocellular carcinoma cells. A: Wound healing assay; B: Transwell migration and Matrigel invasion assay demonstrated that developing brain homeobox 2 (Dbx2) promoted cell migration and invasion. The effects were reversed after Dbx2 knockdown; C: Effect of Dbx2 on expression of several epithelial-mesenchymal transition related proteins detected by Western blot analysis. ^aP < 0.05; ^bP < 0.01.

Figure 5 Developing brain homeobox 2 activates the Shh-Gli1 signaling pathway in hepatocellular carcinoma. A: Ectopic expression of developing brain homeobox 2 (Dbx2) upregulates the expression of Shh, PTCH1, PTCH2, SUFU, and GLI1 proteins, which are downregulated after Dbx2 knockdown; B: Proposed scheme of molecular basis for gain and loss of function of Dbx2 in HCC according to our results.