Genomic analysis reveals RhoC as a potential marker in hepatocellular carcinoma with poor prognosis

Abstract

Hepatocellular carcinoma (HCC) is one of the most malignant human tumours because of its high incidence of metastasis. The mechanisms underlying the metastasis of HCC, however, remain poorly understood. In this study, we performed cDNA microarray analysis to profile gene expression patterns in two subtypes of HCC, solitary large HCC (SLHCC) and nodular HCC (NHCC), which differ significantly in the incidence of metastasis. Among 668 genes that were differentially expressed, we focused on RhoC, whose expression was significantly decreased in SLHCC compared to NHCC. The expression of RhoC in HCC and pericarcinomatous liver tissues (PCLT) was analysed at both the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. In addition, immunohistochemistry was also performed on 94 cases of HCC with follow-up information. Collectively, our data indicate that the expression of RhoC significantly increased in HCC compared to PCLT; extrahepatic metastatic lesions expressed significantly higher levels of RhoC than the corresponding intrahepatic HCC tissues. There is a highly significant correlation of the RhoC expression levels with tumour vein invasion, number of tumour nodes and the status of differentiation. Significantly, the HCC patients with RhoC-positive expression had shorter survival than those with RhoC-negative expression. Together, our findings suggest a strong correlation between the expression of RhoC and HCC metastasis, implicating RhoC as a potential prognosis marker and therapeutic target for HCC.

Figure 1

(A) Typical staining for positive cytoplastic RhoC expression in an HCC (original magnification × 400). (B) Typical staining for negative cytoplastic RhoC expression in an HCC (original magnification × 400).

Figure 2

Detection of RhoC mRNA by RT–PCR. (A) PCR products were visualised by ethidium bromide staining. Eight selected samples from each group are shown: RhoC (181 bp); β-MG (120 bp); EHML: extrahepatic metastatic lesions; HCC: hepatocellular carcinoma tissues; PCLT: pericarcinomatous liver tissues. (B) The Mann–Whitney test was performed to compare the mRNA expression of RhoC between different groups; EHML showed a significantly higher mRNA expression level than HCC and the same between HCC and PCLT.

Figure 3

Western analysis of RhoC protein. (A) The protein expression levels were obtained on Kodak films and were quantified by densitometry. A total of 10 selected samples were shown. (B) The Mann–Whitney test showed significant differences between EHML and HCC, and also between HCC and PCLT.

Figure 4

Correlation between mRNA and protein expression levels of RhoC in HCC was evaluated by Spearman's correlation coefficient. RhoC Protein levels were directly correlated with the levels of RhoC mRNA in HCC with adjusted r_s=0.735, and two-tailed probability, P<0.001.

Figure 5

Kaplan–Meier survival curves for RhoC-positive expression group (n=58) and RhoC-negative expression group (n=36) based on the results of immunohistochemistry. HCC patients with RhoC-positive expression revealed a significantly poor prognosis than those with RhoC-negative expression (log-rank test, P=0.0031).