Samples were normalized using Significance Analysis of Microarrays (SAM), and differentially expressed genes were identified at a nominal P ≤ 0.05. Unsupervised cluster analysis was performed using Cluster and TreeView programs.2. Only genes with a fold change ≥2 were included in the analyses. Functional classification and network analysis were performed using Ingenuity Pathway Analysis tool (Ingenuity Systems Inc.) and the GeneGo microarray tool. Microarray data from 139 HCC samples[21] were used for the survival

analysis according to the SIRT6 signatures. SIRT6 expression was MK-8669 solubility dmso investigated in a subcontingent of 53 HCC tumor specimens.[22] The Oncomine Cancer Microarray database (http://www.oncomine.org) was used to study gene expression of the SIRT6 signature in human HCC and conduct a meta-analysis for the predictive value of the SIRT6 signature in more than 40 different cancer types. Expression values of tumor samples were log-transformed and median-centered and standard deviation was normalized to one per array before comparison to their normal tissue counterparts as described

recently.[23] Statistical analysis was performed using Student t test or analysis of variance as indicated. P ≤ 0.05 was considered statistically significant. Results are presented as the mean ± SD or mean ± SEM as indicated. Univariate and multivariate analysis were performed using a chi-squared test and PD98059 mouse Cox proportional hazard regression, respectively. For the multivariate analyses, only significant variables with sufficient data points were included. To investigate the relevance of SIRT6 for primary human HCC, we first used publically available gene expression data of liver cancer patients from the Oncomine Cancer Microarray database.[23] A significant reduction of SIRT6 expression was revealed in cirrhotic livers and HCC specimens (P < 0.001) compared with levels observed in noncirrhotic

livers (Fig. 1A). In confirmation of these findings, a down-regulation of SIRT6 in HCC tissues compared with nondiseased normal livers was also observed in around 45% (24/53) using independent gene expression data from our recently published cohort of 53 human 上海皓元医药股份有限公司 HCCs (Fig. 1B, upper panel).[22] Consistently, around 42% (16/38) of the tumor samples showed SIRT6 levels below the median center of the expression data of all samples (normalized expression units < 0) of patient samples analyzed in Fig. 1A (Fig. 1B, lower panel). These data indicate a stepwise reduction of SIRT6 in both premalignant and malignant stages of hepatocarcinogenesis. To investigate the gene expression pattern deregulated by SIRT6 loss, we established a SIRT6 KO gene expression signature. To obtain a hepatocyte-specific transcriptome analysis, we isolated primary mouse hepatocytes from wild-type (WT) and Sirt6-deficient livers at 3 weeks of age.