Loss of Barx1 promotes hepatocellular carcinoma metastasis through up-regulating MGAT5 and MMP9 expression and indicates poor prognosis

Abstract

Metastasis is the major dominant reason for poor prognosis of hepatocellular carcinoma (HCC) after surgical treatment. However, the molecular mechanism of metastasis has not been well characterzied. Here, we report a novel function of Barx homeobox1 (Barx1) in inhibiting HCC invasion and metastasis. Barx1 expression is significantly decreased in human HCC tissues than in adjacent non-tumorous tissues and normal liver tissues. Low Barx1 expression is correlated with higher tumor-nodule-metastasis stage and indicates poor prognosis. Down-regulation of Barx1 promotes HCC migration, invasion and metastasis, whereas up-regulation of Barx1 inhibits HCC migration, invasion and metastasis. Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5 (MGAT5) and matrix metallopeptidase 9 (MMP9) are direct target genes of Barx1. Knockdown of Barx1 up-regulates MGAT5 and MMP9 expression in HCC cells with low metastatic capability, whereas over-expression of Barx1 suppresses their expression in HCC cells with high metastatic capability. Knockdown of both MGAT5 and MMP9 significantly decreases the invasion and metastasis abilities induced by Barx1 knockdown. Barx1 expression is negatively correlated with MGAT5 and MMP9 expression in human HCC tissues. Patients with low expression of Barx1 and high expression of MGAT5 or MMP9 are associated with poorer prognosis. Thus, loss of Barx1 represents a prognostic biomarker in human HCC patients.

Keywords: barx homeobox 1; hepatocellular carcinoma; mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5; matrix metallopeptidase 9; metastasis.

Figure 1. Barx1 is significantly down-regulated in human HCC tissues and low expression of Barx1 indicates poor prognosis

(A) Representative Barx1 expression in adjacent non-tumorous tissues and primary HCC tissues detected by immunohistochemical methods. Scale bars represent 200 μm (low magnification) and 50 μm (high magnification). (B) Comparison of Barx1 expression in primary HCC tissues and adjacent non-tumor tissues. (C) Kaplan-Meier analysis of the correlation between Barx1 expression and overall survival of human HCC patients. (D) Real-time PCR analysis of Barx1 expression in normal liver (n = 10), 40 pairs of HCCs and adjacent non-tumorous tissues. (E) Relative mRNA expression of Barx1 in HCC patient samples with recurrence (n = 10) or without recurrence (n = 10). (F) Relative mRNA expression of Barx1 in HCC patient samples with metastasis (n = 30) or without metastasis (n = 30). (G) Real-time PCR analysis of Barx1 mRNA levels in primary HCC tissues and paired metastatic HCC tissues (n=10). *P < 0.05.

Figure 2. Barx1 inhibits HCC invasion and metastasis

(A) Real-time PCR and Western blotting analysis of Barx1 expression in different HCC cell lines. (B) Western blotting analysis of Barx1 expression in the indicated HCC cells. (C) Transwell assay analysis of the migration and invasion abilities of the indicated HCC cells. Data are represented as mean ± SEM for triplicate experiments. (D) Representative images of migration and invasion of the indicated cell lines. (E-I) In vivo metastasis assays. Four stable cell lines were transplanted into the livers of nude mice. (E) Representative bioluminescent imaging (BLI) of the different groups is shown at 10 weeks following orthotopic implantation. The incidence of lung metastasis (F), overall survival (G), the number of lung metastatic foci (H), and representative H&E staining of lung tissues (I) from the different groups is shown. Scale bars represent 200 μm (low magnification) and 50 μm (high magnification). Data are represented as mean ± SEM. *P < 0.05.

Figure 3. Barx1 inhibits MGAT5 and MMP9 transcription

(A and B) Barx1inhibits MGAT5 and MMP9 expression. After SMMC7721 and HCCLM3 cells were infected with Lenti-shBarx1 or Lenti-Barx1, respectively, the mRNA (A) and protein levels (B) of MGAT5 and MMP9 were detected using real-time PCR and western blot techniques, respectively. (C and E) Barx1 inhibits MGAT5 and MMP9 transcription. The MGAT5 or MMP9 promoter constructs were co-transfected with pCMV-Barx1, and the relative luciferase activity was determined. Deletion and selective mutation analysis identifies Barx1-responsive regions in the MGAT5 and MMP9 promoter. Serially truncated and mutated MGAT5 or MMP9 promoter constructs were co-transfected with pCMV-Barx1, and the relative luciferase activity was determined. (D and F) (Left panel) A ChIP assay demonstrate the direct binding of Barx1 to the MGAT5 (D) and MMP9 (F) promoter in HCC cells. Real-time PCR was performed to detect the amounts of immunoprecipitated products. Data are represented as mean ± SEM for triplicate experiments. (Right panel) Barx1 directly binds to the MGAT5 (D) and MMP9 (F) promoter in primary HCC tissues. The hepatocytes were separated from the primary HCC tissues (n = 6) and normal liver tissues (n = 3). The cells were crosslinked and the chromatin were immunoprecipitated by anti-Barx1 or control IgG antibodies, respectively. Data are represented as mean ± SEM. The y-axis represents the relative enrichment of Barx1 compared to the IgG control. *P < 0.05.

Figure 4. Barx1 inhibits HCC metastasis through the inhibition of MGAT5 and MMP9 expression

(A and B) Following the infection of the SMMC7721-shBarx1 cells with the lentivirus Lenti-shMGAT5 or Lenti-shMMP9, respectively, (A) the protein levels of Barx1, MGAT5, and MMP9 were detected by western blot, and (B) the cell migration and invasion capacities were assessed using transwell assays. Data are represented as mean ± SEM for triplicate experiments. (C) Representative images of migration and invasion of the indicated cell lines. (D-H) In vivo metastatic assay. Cell lines were transplanted into the livers of nude mice. (D) Representative bioluminescent imaging (BLI) of the different groups is shown at 10 weeks following orthotopic implantation. The incidence of lung metastasis (E), overall survival (F), the number of lung metastatic foci (G), and representative H&E staining of lung tissues (H) from the different groups is shown. Scale bars represent 200 μm (low magnification) and 50 μm (high magnification). Data are represented as mean ± SEM. *P < 0.05.

Figure 5. Barx1 expression is inversely correlated with MGAT5 and MMP9 expression in human HCC tissues

(A) Representative immunohistochemical images of Barx1, MGAT5, and MMP9 in HCC tissues. Scale bars represent 200 μm (low magnification) and 50 μm (high magnification). (B) The association between the expression of Barx1 and either MGAT5 or MMP9 in HCC patients. (C and E) Kaplan-Meier analysis of the correlation between MGAT5 or MMP9 expression and overall survival of human HCC patients. (D and F) Kaplan-Meier analysis of concurrent Barx1 and MGAT5 or MMP9 expression with overall survival.