HOXB8 promotes tumor metastasis and the epithelial-mesenchymal transition via ZEB2 targets in gastric cancer

Abstract

Purpose: The homeobox B8 (HOXB8) functions as a sequence-specific transcription factor that is involved in development. Increased expression of this gene is associated with a wide variety of tumor; however, its function in gastric cancer has not been clarified. In the present study, the expression of HOXB8 in gastric cancer tissues and influence of HOXB8 on gastric cancer cellular were evaluated.

Methods: The expression levels of HOXB8 mRNA in human gastric cancer tissues were analyzed through quantitative RT-PCR. To test the role of HOXB8 in gastric cancer metastasis, the cell transwell assay was performed. Microarray, ChIP-qPCR, and Western blot were used to explore the possible mechanism that HOXB8 promotes gastric cancer cells metastasis.

Results: In this study, we found that HOXB8 showed higher expression in metastatic tissues than no-metastatic tissues. Overexpression of HOXB8 can promote gastric cancer cells migration and invasion, while silencing HOXB8 leads to the opposite results. Overexpression of HOXB8 also increases the rate of metastasis in NCI-N87 mice, while silencing HOXB8 has the opposite results. Furthermore, HOXB8 promotes epithelial-mesenchymal transformation of AGS cells. We also found that ZEB2 can interact with HOXB8 and may be a downstream factor of HOXB8 by using microarray. Knockdown of ZEB2 can inhibit HOXB8-induced migration and invasion capacity, as well as the epithelial-mesenchymal transformation in gastric cancer cells.

Conclusions: The results showed that HOXB8 plays an important role in the development and metastasis of gastric carcinoma.

Keywords: Epithelial–mesenchymal transition; Gastric cancer; HOXB8; ZEB2.

Fig. 1

HOXB8 is overexpressed in gastric cancer tissues and correlated with metastasis. a HOXB8 expression in gastric cancer tissues was analyzed by qRT-PCR. b HOXB8 expression in no-metastatic and metastatic gastric cancer tissues was analyzed and compared. P < 0.001 in panel B based on the Student’s t test. Error bars SD

Fig. 2

HOXD9 expression is correlated with metastasis of gastric cancer cells. a HOXB8 expression in gastric cancer cell lines was analyzed by Western blotting. b Quantitative chart of HOXB8 protein level in gastric cancer cell lines. c HOXB8 expression in gastric cancer cell lines was analyzed by qRT-PCR. P < 0.001 in b and c based on the Student’s t test. Error bars SD

Fig. 3

Silencing HOXB8 in gastric cancer cells decreases the migration and invasion capacity of gastric cancer cells. a Western Blot analysis of HOXB8 levels in the NCI-N87 established cell lines. b qRT-PCR analysis of HOXB8 levels in the NCI-N87 established cell lines. c Gastric cancer cells with silent expression of HOXB8 possessed less invaded abilities in transwell and Matrigel assay. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 4

Silencing HOXB8 in NCI-N87 cell decreases the rate of tumor metastasis in vivo. a Less number of mice with distant metastasis was found in mouse injected with shHOX8-NCI-N87 cells. b, c Less metastasis foci in gastric cancer cells were counted in each mouse injected with shHOX8-NCI-N87 cells. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 5

Overexpression of HOXB8 in gastric cancer cells increases the migration and invasion capacity of gastric cancer cells. a Western Blot analysis of HOXB8 levels in the AGS established cell lines. b qRT-PCR analysis of HOXB8 levels in the AGS established cell lines. c Gastric cancer cells with high expression of HOXB8 possessed stronger invaded abilities in transwell and Matrigel assay. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 6

Overexpression of HOXB8 in AGS cell increases the rate of tumor metastasis in vivo. a More number of mice with distant metastasis was found in mouse injected with AGS-HOXB8 cells. b, c More metastasis foci in gastric cancer cells were counted in each mouse injected with AGS-HOXB8 cells. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 7

Silencing HOXB8 in NCI-N87 inhibits epithelial–mesenchymal transition. a Western blot analysis showed that silencing HOXB8 causes the epithelial cell markers (E-cadherin and α-catenin) upregulated and mesenchymal cell markers (N-cadherin, fibronectin, and vimentin) downregulated. b qRT-PCR analysis showed that silencing HOXB8 causes the epithelial cell markers upregulated and mesenchymal cell markers downregulated. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 8

Overexpression of HOXB8 in AGS promotes epithelial–mesenchymal transition. a Western blot analysis showed that silencing HOXB8 causes the epithelial cell markers (E-cadherin and α-catenin) downregulated and mesenchymal cell markers (N-cadherin, fibronectin, and vimentin) upregulated. b qRT-PCR analysis showed that silencing HOXB8 causes the epithelial cell markers downregulated and mesenchymal cell markers upregulated. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 9

HOXB8 regulates the expression level of ZEB2. a Clustering of the genes differentially expressed after silencing HOXB8. b The enrichment scores of differential expressing genes in HOXB8 silencing cell line

Fig. 10

ZEB2 expression was affected by HOXB8. The ZEB2 expression levels in HOXB8 silencing cell line were assayed by Western blot (a) and qRT-PCR (b). Detecting ZEB2 expression levels in HOXB8 overexpression cell line by using Western blot (c) and qRT-PCR (d). e ZEB2 expression was positively correlated with HOXB8 expression in gastric cancer tissue arrays. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 11

HOXB8 binding to the promoter of ZEB2 were assayed by ChIP-qPCR. a Regulation of ZEB2 gene promoter activity by knocking down of HOXB8 expression in NCI-N87 cells was analyzed by luciferase assay in NCI-N87 cells. b Regulation of ZEB2 gene promoter activity overexpression of HOXB8 in AGS cells was analyzed by luciferase assay. c Schematic diagram of ZEB2 promoter region. d qChIP was performed in HOXB8 silencing cell lines. f qChIP was performed in HOXB8 overexpression cell lines. e, g IgG was used as negative control. P < 0.01 is based on the Student’s t test. Error bars SD

Fig. 12

Silencing ZEB2 in AGS-HOXB8 cells decreases the migration and invasion capacity of gastric cancer cells. a Western Blot analysis of ZEB2 levels in the established AGS-HOXB8 cells. b qRT-PCR analysis of ZEB2 levels in the established AGS-HOXB8 cells. c Silencing ZEB2 reverses the HOXB8-induced migration and invasion in AGS-HOXB8 cells. d Silencing ZEB2 reverses the HOXB8-induced EMT markers change in AGS-HOXB8 cells. P < 0.01 is based on the Student’s t test. Error bars SD