Association of TIP30 expression and prognosis of hepatocellular carcinoma in patients with HBV infection

Abstract

Altered expression of TIP30, a tumor suppressor, has been observed in many cancers. In this study, we have evaluated the expression of TIP30 in the tissues of 209 hepatocellular carcinomas (HCC) and their adjacent tissues by using a high-density tissue microarray, and analyzed its correlation with the clinical pathological parameters of the patients. The results revealed negative or weak expression of TIP30 in 43.5% (91/209) of the HCC tissues, and in only 27% (56/209) of the adjacent tissues. The expression level of TIP30 in HCC was inversely correlated with serum alpha-fetoprotein (AFP) levels, HBV infection, and tumor differentiation. Multivariate analysis for survival indicated that serum HBV infection was the most significant predictor of poor prognosis in HCC (P = 0.0023), and TIP30 expression and tumor differentiation were also independent indicators in this respect (P = 0.0364 and P = 0.0397, respectively). Patients with medium or high expression levels of TIP30 (TIP30(++/+++) ) had a better 5-year overall survival rate than those with low/negative (TIP30(+/-) ) expression (P < 0.001). TIP30(+/-/) HBV(+) patients had the worst 5-year overall survival rate, whereas TIP30(++/+++) /HBV(-) patients had the best. To further explore the correlation between TIP30 and HBV infection in HCC, HBV(+) hepatoblastoma cell-line HepG2 2.2.15 and HCC cell-line Hep3B were used. Upon silencing of HBV, we observed an upregulation of TIP30 and decreased cell proliferation. In the in vivo studies, we found that the mice inoculated with HepG2 2.2.15 cells with HBV silencing had a prolonged tumor latency and a longer life span, as compared to the control mice inoculated with untreated control cells. In conclusion, the results suggest that downregulation of TIP30 may result from HBV infection, and subsequently promotes the progression of HCC.

Keywords: HBV; Survival; TIP30; hepatocellular carcinomas; tumor suppressor.

Figure 1

Kaplan–Meier estimates of survival in patients with hepatocellular carcinoma. (A) Patients with TIP30 weak/negative (TIP30^+/−) expression (n = 91) had a worse 5‐year overall survival rate (OS) than those with medium or high TIP30 expression (TIP30^++/+++) (n = 118) (P = 0.01). Median OS was 21.8 months versus 29.2 months; (B) Patients without HBV infection (HBV⁻, n = 48) had a better 5‐year OS than those with HBV infection (HBV⁺, n = 161) (P = 0.009). Median OS was 36.3 months versus 21.4 months; (C) Patients in the TIP30^++/+++/HBV⁻ group had the best 5‐year OS than those in the TIP30^++/+++/HBV⁺, TIP30^+/−/HBV⁻, and TIP30^+/−/HBV⁺ groups (P = 0.001). Median OS was 40.9 months, 30.7 months, 29.0 months, and 21.6 months, respectively.

Figure 2

The effect of HBV siRNAs on HBV gene expression. HBsAg concentrations in the culture media of HepG2 2.2.15 (A) and Hep3B cells (B) were measured after transfection with HBV PA siRNA–oligofectamine and controls. *P < 0.05, **P < 0.01 versus random dsRNA + oligofectamine.

Figure 3

The TIP30 expression of HepG2 2.2.15 and Hep3B cells before and after HBV silencing with siRNA. Western blot results showed that TIP30 expression was detected 24 h after HBV silencing with siRNA, and peaked at 48 h.

Figure 4

Cell growth assay of HepG2 2.2.15 (A) and Hep3B cells (B) transfected with HBVPA siRNA–oligofectamine. After HBV was interfered, both cells grew at a much slower rate than the cells transfected with random dsRNA–oligofectamine complex. *P < 0.05,**P < 0.01 versus random dsRNA +oligofectamine.

Figure 5

Growth assay of HepG2 2.2.15 and Hep3B cells transfected with HBVPA siRNA–oligofectamine in nude mice. HepG2 2.2.15 (A) and Hep3B (B) cells transfected with HBVPA siRNA–oligofectamine grew significantly slower in nude mice as compared to the control cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated cells.

Figure 6

Survival curve of mice. (A) Mice were injected s.c. with 2 × 10⁶ HBV siRNA‐transfected HepG2 2.2.15 cells or with wild‐type HepG2 2.2.15 cells; (B) Mice were injected s.c. with 2 × 10⁶ HBV siRNA‐transfected Hep3B cells or with wild‐type Hep3B cells. Mice injected with HBV siRNA‐transfected cells had a longer life span than those injected with wild‐type cells. Data were evaluated using a Kaplan–Meier survival curve and log‐rank test.