The expression of TIMP3, a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. mature miR-181b NB001 were augmented upon exposure of hepatic cells to TGF and were significantly reduced by siRNA-mediated depletion of Smad4, demonstrating the involvement of TGF signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced MMP2 NB001 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of HCC cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells innudemice. miR-181b also enhanced resistance of HCC cells to the anti-cancer drug doxorubicin. Based on these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis. Keywords:miR-181b, TIMP3, hepatocarcinogenesis, CDAA diet, Doxorubicin, hepatocullular carcinoma == INTRODUCTION == Different strategies have been used to treat hepatocellular carcinoma (HCC) which is the fifth most prevalent cancer and the third leading cause of cancer death in the world (El-Seraget al., 2007). Despite these efforts, the survival rate has been dismal probably due the late stage diagnosis of this cancer. Like other cancers, HCC is the result of NB001 a complex, multi-step process associated with various genetic and epigenetic changes. Many factors are involved in the development of HCC, which include virus infection, chronic alcohol abuse, obesity, diabetes, and nonalcoholic fatty liver diseases (NAFLD), which is increasing in Western world leading to nonalcoholic steatohepatosis (NASH) and HCC (Aravalliet al., 2008). While the underlying molecular mechanisms involved in the pathogenesis of HBV or HCV infection have been explored in great CYCE2 detail, the pathophysiology and the detailed mechanisms of the initiation and progression of NASH-associated HCC have not been completely understood. Most of the usual chemotherapeutic strategies using different drugs have met with limited success to treat liver cancer. Recently lots of interests have been focused on microRNA (miRNA) mimetics and anti-sense microRNA as potential therapeutics for hepatocellular carcinoma due to their stability and predominant uptake by the liver. MicroRNAs are endogenous, short (20~22-nucleotide), non-coding RNAs that regulate gene expression posttranscriptionally by blocking translation at initiation or post-initiation steps, inducing mRNA deadenylation and decay (Carthewet al., 2009;Eulalioet al., 2008). The genes encoding miRNAs are transcribed in the nucleus predominantly by RNA polymerase II into primary miRNAs (pri-miRNAs), that are processed into ~70-nucleotide precursor miRNAs (pre-miRNAs) by Drosha-DGCR8 microprocessor complex (Kimet al., 2009). Pre-miRNAs are then transported into the cytoplasm by Exportin 5 and further processed into mature miRNAs by Dicer and cofactors (Winteret al., 2009). Mature miRNAs are incorporated into miRNA-induced gene silencing NB001 complex (miRISC) and guide miRISC to specific target mRNAs to exert biological functions (Bartel, 2009). MicroRNAs are involved in various biological processes, such as development and differentiation, immune response, metabolism, cell proliferation and apoptosis (Schickelet al., 2008). The expression profile of miRNAs is altered in disease states and may be involved in the initiation and progression of many types of cancers by targeting classic oncogenes or tumor suppressors (Visoneet al., 2009). miRNA expression profiling is emerging as a potentially powerful tool in the diagnosis and prognosis of diseases. The role of miRNAs in the development and progression of hepatocellular carcinoma (HCC) is emerging only recently. To elucidate the role of specific miRNAs in the initiation and progression of hepatocarcinogenesis, we used a mouse model to identify temporal changes in miRNA expression at early stage of hepatocarcinogenesis. In this model, mice fed choline deficient and amino acid defined (CDAA) diet develop nonalcoholic steatohepatitis (NASH) at early stages leading to preneoplastic nodules after 65 weeks, and hepatocellular adenomas and carcinomas after 84 weeks (Dendaet al., 2002). Using this diet model, we have identified several miRs that are dysregulated during hepatocarcinogenesis (Wanget al., 2009). Here, we focused on the role of one of these miRNAs, miR-181b, in hepatocarcinogenesis, and elucidated the role of TGF signaling pathway in its induction. == Materials and Methods == == Mice and diet == The animal model and the dietary regimen to induce HCC are identical to those described previously (Wanget al., 2009). == Real-time reverse-transcription polymerase chain reaction (RT-PCR) == The TaqMan miRNA Assay (Applied Biosystems, Foster City, CA, USA) was used to determine expression of mature miRNAs expression according to manufacturer’s instructions and the expression.