6D). To further determine the correlation of HNF4α and miR-134 in HCC, we examined their expression profiles in the DEN-induced HCC rat model (n = 4 at each indicated Mitomycin C clinical trial timepoint). In agreement with our previous study,[8] HNF4α expression decreased gradually after DEN administration. Interestingly, the transcript level of miR-134 (pri-miR134) was also suppressed in the process of hepatocarcinogenesis (Fig. 7A). A striking positive correlation between HNF4α and pri-miR-134 levels was observed in DEN-treated rat liver (Fig. 7B). We then analyzed the

association of HNF4α and pri-miR-134 expression in human HCC samples (n = 71). As compared with their surrounding noncancerous tissues, 63% (45/71) and 70% (50/71) of the HCC tissues showed lower levels of HNF4α and pri-miR-134, respectively check details (Fig. 7C). Reduced pri-miR-134 expression was more frequent in HCCs with lower HNF4α levels relative to those with

intermediate and high HNF4α levels (89% versus 38%; Fig. 7D). The correlation was particularly apparent in HCC subjects with alpha-fetoprotein (AFP) levels over 1,000 μg/L (r = 0.6241, P = 0.0003, n = 29; Fig. 7E). The clinicopathological significance of pri-miR-134 levels in the above 71 patients with HCC was further analyzed. The median value of pri-miR-134 levels in HCC tissues was chosen as the cutoff point; 49.3% of HCCs (35/71) had low-level expression of pri-miR-134, and 50.7% of HCCs (36/71) had high-level expression of pri-miR-134 (Table 1). The low-level expression of pri-miR-134 in HCCs was associated with more aggressive pathological features, including liver cirrhosis (P = 0.0127), high levels of AFP (P = 0.0142), large tumor size (P = 0.0271), advanced tumor stage (P = 0.0051), presence of tumor microsatellites (P = 0.0434), and absence of tumor encapsulation (P = 0.0013) (Table 1). HNF4α is a transcription factor that plays a key role in hepatocyte differentiation

and in the maintenance of hepatic function. It is well established that the miRNAs at the DLK1-DIO3 imprinting locus are critical for the differentiation of stem cells and for the development of the mouse embryo.[14, 15, 32] The DLK1-DIO3 miRNA cluster is up-regulated in c-MET mouse liver tumors and in MRIP a subgroup of HCC patients[26]; however, the expression status and function of this miRNA cluster in human HCC are largely unknown. In the current study, we demonstrated that the HNF4α-regulated miR-379-656 cluster in the DLK1-DIO3 region is suppressed in the majority of HCC tumor tissues. Experiments in cultured HCC cells confirmed a suppressive effect of the miR-379-656 cluster on malignant phenotypes. The DLK1-DIO3 imprinted locus contains three paternally expressed protein-coding genes and several maternally expressed noncoding RNA genes (MEGs).