14 Total cell RNAs from HCV-infected and mock-infected cells, 6 days postinfection, were hybridized to microchips affixed with the sequences of approximately 350 known human miRNAs. As shown in Fig. 1, the levels of miRNA expression in primary hepatocytes infected with the three major strains

of HCV were accentuated to various degrees. Consistently, cells infected with the HCV strains showed induced expression of miR-141, miR-200a, and Z-VAD-FMK nmr miR-200b and miR-200c to a lesser degree (Fig. 2; Supporting Information Table 1). miR-141 levels increased concomitantly with HCV infection. We chose miR-141 for further studies because it was consistently the most enriched miRNAs up-regulated in HCV-infected cells. The levels of miR-141 that are consistently accentuated in HCV infection is summarized in (Table 1). Previous studies have analyzed alterations in miRNA in liver cancer tissues compared with adjacent normal tissues. For the most part, miRNA expression appears to be accentuated in HCC, with down-regulated Neratinib miRNAs in HCC being rare.15, 16 Earlier studies of HCV-infected hepatocytes indicated that virus replication depends on host cell miR-12217–19

or the cellular RNA interference machinery in general.20 Because both miR-141 and miR-200a share a target sequence, we chose to study the biological effects of miR-141 up-regulated during HCV infection. We focused next on validating the target of miR-141 that is induced in HCV-infected cells. miRNAs act upon their mRNA targets by way of imperfect base pair complementarity, and subsequently modulate the translation of the mRNA. The most important determinant for targeting efficacy is the strength of the interaction at the 6- to 7-bp seed sequence

at the 5′ end of the miRNA.21–24 We used bioinformatics methods to determine a small number of candidate mRNA targets for the four miRNAs (miR-141, miR-200a, miR-200b, miR-200c) based on the following criteria: (1) strength of the miRNA-target duplex, particularly at the seed position; (2) combinatorial effects Cobimetinib in vitro of multiple binding sites of the same miRNA or of multiple miRNAs targeting the same gene; and (3) conservation of miRNA target sites in multiple related species, which increases their likelihood to be functional.21, 25 Using Pictar software,26 we searched for genes containing multiple miR-141, miR-200a, miR-200b, and miR-200c target sites that are conserved in humans, chimpanzees, mice, rats, and dogs. Whereas Pictar reported 100-200 target genes for each of the miRNAs individually, the combinatorial search revealed a more specific set of 65 potential genes (Supporting Information Table 2). We narrowed down the set of targets further based on the functional characterization of genes, prioritizing those likely associated with HCV infection and liver disease. DLC-1 (GenBank accession no.