Bmi-1 gene is upregulated in early-stage hepatocellular carcinoma and correlates with ATP-binding cassette transporter B1 expression

Abstract

Overexpression of "stemness gene"Bmi-1 has been identified in some solid tumors. We investigated Bmi-1 expression in hepatocellular carcinoma (HCC) and ATP-binding cassette transporter B1 (ABCB1) as a new potential target for Bmi-1. Bmi-1 was highly expressed in HCC cell lines and the most well differentiated cell line, KIM-1, showed the highest expression. Immunohistochemical, immunocytochemical, and immunoelectron microscopic analysis showed the Bmi-1 protein as having a high intensity of small dots within the nucleus which reflected concentrated sites of Bmi-1 repressive activity. Clear "dot-pattern" staining was observed in 24 of 37 (65%) well differentiated HCC (including 13 of 21 early nodules [62%]), in 32 of 71 (45%) moderately differentiated HCC, and 7 of 14 (50%) poorly differentiated HCC. A similar expression was not observed in non-cancerous background regions. High Bmi-1 expression was observed in the early and well differentiated HCC. Furthermore, overexpression and suppression of Bmi-1 was followed by a respective increase and decrease in ABCB1 expression. As with Bmi-1, high ABCB1 expression was also observed in the early and well differentiated HCC. A strong correlation between ABCB1 and Bmi-1 mRNA expression was seen in HCC cell lines and clinical samples (Pearson's correlation coefficient 0.95 and 0.90, respectively). The Bmi-1 gene is upregulated in HCC, and in particular is highly expressed in early and well differentiated HCC. The fact that this expression correlated with that of ABCB1 suggests a new regulation target for Bmi-1, and gives new insight into early hepatocarcinogenesis mechanisms and potential targets for future HCC treatment.

Figure 1

Bmi‐1 expression in hepatocellular carcinoma (HCC) cell lines. (A) Quantitative real‐time‐ PCR and Western blot of Bmi‐1 in HCC cell lines. Bmi‐1 is significantly expressed in the KIM‐1 cell line compared with other cell lines. Nuclear fraction proteins were used in the Western blot analysis. Immunohistochemistry (B) and immunocytochemistry (C) of KIM‐1 cells. Bmi‐1 was diffusely distributed intra‐nuclearly (a, DAPI: blue; b, anti‐Bmi‐1: red; c, merged). (D) Immunoelectron micrograph of KIM‐1. Bmi‐1 particles are shown as small black dots inside the nucleus.

Figure 2

Bmi‐1 expression in hepatocellular carcinoma (HCC) clinical samples. (A) Immunostaining of Bmi‐1 in moderately differentiated HCC. Magnification, ×200. A clear dot‐pattern of Bmi‐1 was distributed diffusely in the tumor region. (B) Boundary region of well differentiated HCC (a, H&E stain; b, corresponding Bmi‐1 staining, magnification ×100; c, magnification ×200). Bmi‐1 expression was observed in the tumor region but not in surrounding liver tissue. Black arrows outline the border between the non‐cancerous background region (N) and the tumor region (T). (C) Bmi‐1 mRNA expression levels in HCC clinical cases. The relative mRNA expression levels in tumor tissues (black bar, T) and corresponding non‐cancerous, background liver tissues (gray bar, N) (left panel). High Bmi‐1 expression was observed in well differentiated HCC. The average expression level of Bmi‐1 was significantly higher in tumor tissues than in non‐cancerous, background liver tissues (2.23 vs 0.86; *P = 0.002) (right panel).

Figure 3

Overexpression and silencing of Bmi‐1 expression affected ATP‐binding cassette transporter B1 (ABCB1) expression in primary fetal hepatocytes and a hepatocellular carcinoma (HCC) cell line. (A) Bmi‐1 overexpression in primary fetal hepatocytes resulted in increased ABCB1 expression, compared with the mock‐transduced control (*P < 0.01; ***P = 0.038). (B) Silencing of Bmi‐1 expression by two different siRNAs (#1 and #2) in KIM‐1 cells was followed by a decrease in ABCB1 expression (*P < 0.01; **P = 0.08). Error bars were derived from three independent experiments.

Figure 4

Correlation and immunostaining of Bmi‐1 and ATP‐binding cassette transporter B1 (ABCB1) expression in hepatocellular carcinoma (HCC). (A and B) Evaluation of Bmi‐1 and ABCB1 mRNA expression in HCC cell lines and HCC clinical samples. A strong correlation between Bmi‐1 and ABCB1 expression was observed in HCC cell lines and clinical samples by the Pearson’s correlation coefficient test (0.95, P = 0.01; and 0.90, P < 0.01, respectively) (black column, Bmi‐1; gray column, ABCB1). (c) Bmi‐1 and ABCB1 expression in early, moderately, and poorly differentiated HCC (magnification, ×200). Clear staining of Bmi‐1 “dot‐pattern” (scored as 2+), and a canalicular and cytoplasmic ABCB1 staining pattern (scored as 2+), was observed in well differentiated HCC. Bmi‐1 expression appeared weaker (scored as 1+), and only a canalicular staining pattern of ABCB1 (scored as 1+), was seen in moderately differentiated HCC. No dot‐pattern of Bmi‐1 and an absence of ABCB1 staining were observed in poorly differentiated HCC (scored as negative). Both Bmi‐1 and ABCB1 expression decreased with the progression of HCC, suggesting their correlated expression.