9%) of 32 HCCs tested were weak or negative-stained. Therefore, it seems that HDAC6 is down-regulated during hepatocarcinogenesis. To generalize our finding, we recapitulated HDAC6 gene expression from the large cohorts of HCC patients that are available from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession numbers GSE14520 and GSE25097) and shown as scatterplots. Consistently, HDAC6 gene expression was significantly down-regulated in two different HCC cohorts (Fig. 1B,C). Decreased expression of HDAC6 protein was confirmed by western immunoblotting
of six randomly selected human HCC tissues paired with N and DN. As expected, HDAC6 was markedly down-regulated in all selected HCCs as compared with normal liver or dysplatic nodule tissues INK 128 manufacturer (Fig. 1D; Supporting Fig. 1C). Furthermore,
endogenous expression of HDAC6 was investigated by northern and western blot analysis in nine different liver cancer cell lines (HepG2, Hep3B, PLC/PRF/5, SNU182, SNU354, SNU368, SNU387, SNU423, and SNU449), which were originally established from HCC or hepatoblastoma. The human liver cancer cell lines exhibited relatively low HDAC6 expression, with a few exceptions (Fig. 1E). These results strongly suggest that HDAC6 is suppressed in HCC. Because the messenger RNA (mRNA) and protein levels of HDAC6 showed down-regulation in overt HCCs, we next assessed the prognostic association HM781-36B research buy learn more of HDAC6 expression in a large cohort of 100 Korean HCC patients.17 First, 1,909 genes with expression patterns that highly correlated with HDAC6 expression were selected for cluster analysis
(P < 0.001, r > 0.4 or r < −0.4), and shown as heatmaps (Fig. 2A). Patients were then divided into the following two groups: HDAC6 High cluster and HDAC6 Low cluster. The Kaplan-Meier survival curves of patients with HCC indicated that the 5-year overall survival (OS) rate of HCC patients with low HDAC6 expression (50.9%) was significantly lower than that of HCC patients with high HDAC6 expression (69.4%, P < 0.05; Fig. 2B). The disease-free survival (DFS) rate of HCC patients with low HDAC6 expression (27.5%) was also significantly lower than that of HCC patients with high HDAC6 expression (44.9%, P < 0.05; Fig. 2C). In addition, the recurrence-free survival (RFS) rate of HCC patients with low HDAC6 expression (35.3%) was significantly lower than that of HCC patients with high HDAC6 expression (53.1%, P < 0.05; Fig. 2D). These results demonstrated that HDAC6 expression is strongly associated with prognosis in HCC patients. To better understand the molecular consequences of ectopic overexpression of HDAC6 in hepatocarcinogenesis, full-length human HDAC6 cDNA (pcDNA_HDAC6) was constructed and transiently transfected into Hep3B cells and cell viability and MTT cell proliferation assays were performed. The functional activity of HDAC6 was confirmed by detecting the hypoacetylation of α-tubulin (Fig.