Overexpression of RHEB is associated with metastasis and poor prognosis in hepatocellular carcinoma

Abstract

Aberrant expression of Ras homolog enriched in brain (RHEB) has been observed in a variety of cancer tissues and is closely associated with clinicopathological features. However, the expression profile of RHEB in patients with hepatocellular carcinoma (HCC) and its clinical signature with underlying mechanisms have not been explored thus far. To analyze the association between RHEB expression and clinicopathological features, the RHEB expression levels were determined in the present study using gene microarrays, immunohistochemistry and western blotting in 60 liver cancer tissues and 35 normal liver tissues. Downregulation of RHEB expression in liver cancer cell lines was achieved by RNA interfering technology to explore its biological function in HCC. RHEB expression was high in liver cancer tissues, with an increase of 2.00±0.19-fold compared with normal tissues and of 2.00±0.27-fold compared with adjacent non-cancer tissues. RHEB expression increased along with the clinical staging of HCC, and the overall survival and mortality of patients were closely correlated to RHEB levels, micro-vascular invasion, hepatitis B virus-DNA titer, tumor differentiation and pathological satellites (P<0.05). After knocking down RHEB in SMMC-7721 cells, the growth of liver cancer cells was significantly reduced. The majority of cells were blocked in S-phase, and their colony-forming and proliferating abilities significantly decreased (P<0.05). In vivo, upon downregulation of RHEB expression, the tumorigenic ability of HCC significantly decreased (P<0.05). These data suggest that RHEB expression is a significant prognostic factor and may be important in HCC cell growth. The present study highlights the importance of RHEB as a novel prognostic marker of HCC.

Keywords: HCC; RHEB; RNA interference; gene microarray; metastasis; prognosis.

Figure 1.

RHEB expression in liver cancer cell lines and tissues. (A) RHEB was overexpressed in Hep3B, SMMC-7721, HepG2 and MHCC-97-H cells, but not in MHCC-97-L cells, as demonstrated by western blot analysis. (B) RHEB expression in liver cancer relative to the control gene GAPDH (5.00±0.34-fold) was upregulated compared with that in adjacent tissues (2.00±0.27-fold) and normal tissues (2.00±0.19-fold), as evaluated by gene microarray analysis. (C) RHEB messenger RNA level in liver cancer samples was upregulated compared with that in non-cancerous liver samples, as demonstrated by reverse transcription-quantitative polymerase chain reaction analysis. (D) RHEB was overexpressed in liver cancer tissues, but little or no expression was detected in normal liver tissues, as analyzed by immunohistochemistry. Magnification, ×200. RHEB, Ras homolog enriched in brain; mRNA, messenger RNA.

Figure 2.

Correlation analysis between RHEB expression and clinicopathological data of patients with HCC. (A) The differences in RHEB expression between different tumor stages in liver cancer tissues was significant (P=0.020). (B and C) Correlation analysis between different clinicopathological data and (B) survival time and (C) postoperative recurrence of patients with HCC. RHEB, Ras homolog enriched in brain; IOD, integral optical density; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; HBV, hepatitis B virus.

Figure 3.

Influence of silencing RHEB on the growth of SMMC-7721 cells. (A) RHEB expression in SMMC-7721 cells treated with RHEB-shRNA according to by western blot analysis. (B) Variation in interference efficiency in SMMC-7721 cells treated with different shRNAs. (C and D) Changes in cell proliferation ability upon treatment with shRHEB were reported as (C) cell count and (D) cell count/fold. RHEB, Ras homolog enriched in brain; mRNA, messenger RNA; scr, scramble; shRNA, small hairpin RNA.

Figure 4.

Influence of silencing RHEB on the biological behavior of the liver cancer cell line SMMC-7721. (A) Cell cycle changes of SMMC-7721 cells with low expression of RHEB were analyzed by flow cytometry. (B) Comparison of the cell cycle changes in SMMC-7721 cells treated with different shRNAs. (C) Analysis of soft agar colony-formation ability of SMMC-7721 cells with downregulated RHEB expression. The experiments were performed in triplicate, and representative images are shown. RHEB, Ras homolog enriched in brain; scr, scramble; FL2-A, fluorescence 2-area; shRNA, small hairpin RNA.

Figure 5.

Influence of RHEB silencing on the biological behavior of nude mice. Influence of low RHEB expression on (A) tumor growth and (B) nude mice tumorigenic ability. RHEB, Ras homolog enriched in brain; scr, scramble; shRNA, small hairpin RNA.