Univariate analysis of SIRT3 expression and clinicopathologic variables in 248 patients with primary hepatocellular carcinoma (log-rank test).

a<p>Mean age;</p><p>NR, not reached; HbsAg, hepatitis B surface antigen; AFP, alpha-fetoprotein.</p

Low SIRT3 Expression Correlates with Poor Differentiation and Unfavorable Prognosis in Primary Hepatocellular Carcinoma

<div><p>SIRT3, a mitochondrial sirtuin belonging to nicotinamide adenine nucleotide (NAD) dependent deacetylases, is implicated in metabolism, longevity and carcinogenesis. SIRT3 expression and its significance in hepatocellular carcinoma (HCC) remain largely unclear. In this study, we demonstrated that SIRT3 expression in HCC tissue was much lower than that in paracarcinoma tissue, at both mRNA and protein levels. The cutoff value for low SIRT3 expression in HCC was defined according to receiver operating characteristic curve (ROC) analysis. As disclosed by immunohistochemistry (IHC) results, low SIRT3 expression was present in 67.3% (167/248) of HCC cases. Furthermore, low expression of SIRT3 was significantly correlated to differentiation (<em>P</em> = 0.013), clinical stage (<em>P</em> = 0.005), serum AFP level (<em>P</em><0.01), tumor multiplicity (<em>P</em> = 0.026) and relapse (<em>P</em> = 0.028). Moreover, Kaplan-Meier analysis indicated that low SIRT3 expression associated with unfavorable overall survival (<em>P</em><0.01) and recurrence-free survival (<em>P</em> = 0.004). The prognostic impact of SIRT3 was further confirmed by stratified survival analysis. Importantly, multivariate analysis revealed that low SIRT3 expression was an independent poor prognostic marker for overall survival (Hazard Ratio (HR) 0.555, 95% confidence interval (95% CI) 0.344–0.897, <em>P</em> = 0.016). Collectively, we conclude that SIRT3 is decreased in HCC and is a novel unfavorable marker for prognosis of patients with this fatal disease.</p> </div

Protein expression patterns of SIRT3 in HCC tissues by IHC.

<p>The immunoreactivities were primarily observed in cytoplasm within tumor and normal liver cells. The micrographs showed negative (<b>A</b>), low (<b>C</b>), and high (<b>E</b>) expression of SIRT3 in HCC tissues. The relevant expressions of SIRT3 in corresponding adjacent normal liver tissues of cases in <b>A</b>, <b>C</b> and <b>E</b> were shown in <b>B</b>, <b>D</b> and <b>F</b>, respectively. (Left panel: magnification ×100; Right panel: magnification ×400.)</p

Reverse association of SIRT3 expression in HCC tissue with tumor differentiation.

<p><b>A.</b> Representative micrographic images were shown to present the correlation between SIRT3 expression in HCC and differentiation. <b>B.</b> Percentages of high SIRT3 expressions in differentiated HCC were indicated by histogram.</p

Expression of Fibulin-3 in HCC tissues by IHC.

<p>Micrographs showed weak (A), moderate (B), and strong (C) staining of Fibulin-3 in HCC, as well as low (D) and high (E) expression of Fibulin-3 in normal liver tissues. (Left panel: magnification ×100; Right panel: magnification ×400.) F. Reproducibility of the measurement in all 255 patients was calculated using the Wilcoxon matched paired test.</p

Correlation of Fibulin-3 expression with overall survival in morphologic and pathological HCC subgroups.

<p>Survival analyses were performed in subgroups divided by the factors that contribute to poorer outcomes of HCC patients, using Kaplan-Meier survival analysis (log-rank test).</p

Correlation of low SIRT3 expression in HCC tissue with unfavorable overall survival and recurrence-free survival.

<p>Probabilities of overall survival <b>(A)</b> and recurrence-free survival <b>(B)</b> of total 248 HCC patients were analyzed by Kaplan-Meier survival analysis (log-rank test).</p

Correlation between the clinicopathologic variables and SIRT3 expression in HCC.

a<p>Chi-square test;</p>b<p>Mean age;</p><p>AFP, alpha-fetoprotein; HBsAg, hepatitis B surface antigen.</p

Relation of SIRT3 expression with overall survival in morphologic and pathological HCC subgroups.

<p>Survival analysis was performed in subgroups according to the factors that are attributed to worse outcome of HCC patients, using Kaplan-Meier survival analysis (log-rank test).</p

Expression of SIRT3 in HCC cell lines and tissue samples. A.

<p>mRNA level of SIRT3 in immobilized liver cell line (MiHA) and HCC cell lines was determined by qRT-PCR. Three independent experiments were performed. Data are mean ± SD. <b>B.</b> Representative pattern of SIRT3 protein expressed in cell lines was shown. The ratio of SIRT3/GAPDH was indicated as well. <b>C.</b> mRNA level of SIRT3 in HCC and corresponding adjacent liver tissue was determined in 16 patients. Relative SIRT3 mRNA in HCC tissues was presented. <b>D.</b> Wilcoxon matched paired test revealed the significant alteration of SIRT3 mRNA in tissue samples. <b>E.</b> Expression of SIRT3 protein in 16 paired HCC and adjacent normal liver tissues were examined by western blot. <b>F.</b> Relative intensity of PLK4 normalized to GAPDH was calculated.</p