The suppressive role and aberrent promoter methylation of BTG3 in the progression of hepatocellular carcinoma

Abstract

Background: BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology: The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results: BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion: Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.

Figure 1. BTG3 is down-regulated in clinical paraffin-embedded HCC tissues and a prognostic factor for poor overall survival in HCC patients.

(A) Strong positive expression of BTG3 in adjacent normal livers (×200, ×400). (B) Positive expression of BTG3 in the cirrhotic livers (×100, ×400). (C) Negative expression of BTG3 in HCC tissues compared with strong positive expression in adjacent livers (×200, ×400). (D) Week expression of BTG3 in HCC tissues while strong positive expression in adjacent livers (×200, ×400). (E) Negative expression of BTG3 in HCC tissues with low differentiation (×200, ×400). (F) Weak expression of BTG3 in HCC tissues with high differentiation (×200, ×400). (G) Kaplan-Meier survival analysis of primary HCC patients with high and low BTG3 expressions.

Figure 2. BTG3 is down-regulated in HCC cell lines and fresh HCC tissues.

(A) Real-time RT-PCR analysis of BTG3 in six cell lines. The relative mRNA levels with the use of control LO2 were normalized to 1. (B) Western blotting analysis of BTG3 in six cell lines. Tubulin was shown as a control. (C) Real-time PCR analysis of BTG3 expression in 20 paired HCC tissues. The relative mRNA levels with the use of control normal livers were normalized to 1. (D) Real-time PCR analysis of BTG3 expression in all cases of HCC tissues and adjacent normal livers. (E) Western blotting analyses of BTG3 expression in 8 paired HCC tissues. Tubulin was shown as a control. N=normal mucosa and T=tumor.

Figure 3. Promoter hypermethylation contributes to the transcriptional silencing of BTG3 in HCC.

(A) Real-time PCR analysis of BTG3 expression after 5Aza-C treatment in HepG2 and 7721 cells. The relative mRNA levels with the use of control cells without 5Aza-C treatment were normalized to 1. (B) Western blotting analysis of BTG3 expression in HepG2 and 7721 cells after 5Aza-C treatment. Tubulin was shown as a control. (C) DNA sequencing results of methylation sites in LO2 cells. (D) DNA sequencing results of methylation sites in HepG2 and 97H cells. (E) Ratio of methylation sites of HepG2 or 97H cells was compared with that of LO2 cells.

Figure 4. BTG3 inhibits proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

(A) BTG3 expression in FBX8 over-expressing 7721 and HepG2 cells by Western blot. Tubulin was shown as a control. (B) Effect of ectopic BTG3 on cell proliferation in vitro by MTT assay. (C) Effect of ectopic BTG3 on cell invasion in vitro by using Boyden chambers. (D) Morphological comparison of BTG3-overexpressing cells and control cells penetrating the artificial basement membrane was shown. (E) Effect of ectopic BTG3 on cell cycle transition in vitro by flow cytometry.

Figure 5. BTG3 knockdown promotes proliferation, invasion and G1/S phase transition of HCC cells in vitro.

(A) BTG3 expression in BTG3 depleting 97H and 7701 cells by Western blot. (B) Effect of BTG3 knockdown on cell proliferation in vitro by MTT assay. (C) Effect of BTG3 knockdown on cell invasion in vitro by using Boyden chambers. (D) Morphological comparison of BTG3-depleting cells and control cells penetrating the artificial basement membrane was shown. (E) Effect of BTG3 kncokdown on cell cycle transition in vitro by flow cytometry. (F) Expression levels of Cyclin D1 and p27 in BTG3-overexpressing and depleting cells by Western blot. Tubulin was shown as a control.