10), supporting the hypothesis that the NF-κB pathway is involved in the suppressive effects of miR-370 on HCC. IL-6 is a downstream target gene of NF-κB that plays a crucial role in hepatocarcinogenesis.[4, 5] Previous studies reported that IL-6 inhibited miR-370 through modulation of DNA methylation in human cholangiocarcinoma (CCA).[12, 13] In this study, we confirmed that treatment of HCC cells with IL-6 significantly decreased miR-370 levels, followed by an increase in LIN28A protein (Fig. 6E and Supporting Fig. 11A,B). Furthermore, the methylation inhibitor, 5-aza-2′-deoxycytidine

markedly increased expression of primary miR-370 (Supporting Fig. 11C), suggesting that miR-370 is down-regulated in HCC in an epigenetic manner. These results indicate that a positive feedback loop, consisting of miR-370, LIN28A, RelA/p65, and IL-6, is involved in the progression of HCC mTOR inhibitor (Fig. 6F). We further validated the

roles of miR-370 and LIN28A in the development of HCC by using real-time PCR to examine miR-370 and LIN28A mRNA levels in liver tissues from diethylinitrosamine (DEN)-treated rats, 86 paired primary HCC and adjacent nontumorous liver tissues from primary HCC patients with complete clinical data (excluding the 20 pairs of samples referred to above), and 24 healthy human liver tissue samples. miR-370 expression was significantly down-regulated in cirrhotic liver tissues from rats at week 11 after DEN administration, compared to normal rat livers, and was further reduced in HCC tissues, relative to adjacent fibrotic tissues (Fig. 7A). In contrast, LIN28A mRNA was gradually up-regulated during the development Target Selective Inhibitor Library in vivo of DEN-induced HCC (Fig. 7B). Consistently, miR-370 expression was substantially repressed in the surrounding nontumorous livers from HCC patients, compared to healthy human

livers (median, 0.859 and 0.003, Etofibrate respectively; P < 0.001; Mann-Whitney’s U test), and was further reduced in HCC tissues (Fig. 7C). In contrast, LIN28A mRNA levels were increased 19-fold in nontumorous livers, compared to healthy livers. LIN28A mRNA was only slightly augmented in HCC tissues, relative to surrounding nontumorous tissues (median, 3.59 × 10−4 and 6.08 × 10−4, respectively; Fig. 7D). Correlation studies displayed that miR-370 levels were inversely correlated with LIN28A and IL6 mRNA levels in human HCC specimens (Fig. 7E), and LIN28A expression was positively correlated with IL-6 expression (Fig. 7E). Specifically, low expression of miR-370 was more likely in human HCC specimens with high levels of LIN28A and IL-6 mRNAs, whereas high expression of LIN28A was more likely in human tissues with high IL-6 levels. More interesting, clinicopathologic analysis demonstrated that down-regulation of miR-370 in human HCCs was significantly correlated with aggressive pathologic characteristics, including larger tumor size (P = 0.028), advanced tumor stage (P = 0.