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Nucleolin, as a multifunctional protein, has been demonstrated to play an oncogenic role in human hepatocellular carcinoma (HCC). The aim of this study was to investigate the expression pattern of nucleolin in HCC and determine its correlation with tumor progression and prognosis. Nucleolin expression at both mRNA and protein levels in HCC and adjacent nonneoplastic tissues were respectively detected by quantitative real time polymerase chain reaction (Q-PCR), immunohistochemistry and western blotting. Nucleolin expression, at both mRNA and protein levels, was significantly higher in HCC tissues than in the adjacent nonneoplastic tissues (both P < 0.001). In addition, the elevated nucleolin expression was markedly correlated with advanced tumor stage (P = 0.001), high tumor grade (P = 0.02) and serum AFP level (P = 0.008). Moreover, HCC patients with high nucleolin expression had shorter 5-year disease-free survival and shorter 5-year overall survival than those with low expression (both P < 0.001). Furthermore, the Cox proportional hazards model showed that nucleolin expression was an independent poor prognostic factor for both 5-year disease-free survival (hazards ratio [HR] = 3.696, 95% confidence interval [CI] = 1.662-8.138, P = 0.01) and 5-year overall survival (HR = 3.872, CI = 1.681-8.392, P = 0.01) in HCC. These results showed that the markedly and consistently increasing expression of nucleolin may be associated with aggressive characteristics of HCC, and implied that nucleolin expression may serve as a promising biochemical marker for predicting the clinical outcome of patients with this malignancy. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_175 .