Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.

MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G1 to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH. Conclusion Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration.

Background Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HCC). However, the mechanistic pathways that link both disorders are essentially unknown. Objective Our study was designed to investigate the role of microRNA-21 in the pathogenesis of NAFLD and its potential involvement in HCC. Methods Wildtype mice maintained on a high fat diet (HFD) received tail vein injections of microRNA-21-anti-sense oligonucleotide (ASO) or miR-21 mismatched ASO for 4 or 8 weeks. Livers were collected after that time period for lipid content and gene expression analysis. Human hepatoma HepG2 cells incubated with oleate were used to study the role of miR-21 in lipogenesis and analysed with Nile-Red staining. microRNA-21 function in carcinogenesis was determined by soft-agar colony formation, cell cycle analysis and xenograft tumour assay using HepG2 cells. Results The expression of microRNA-21 was increased in the livers of HFD-treated mice and human HepG2 cells incubated with fatty acid. MicroRNA-21 knockdown in those mice and HepG2 cells impaired lipid accumulation and growth of xenograft tumour. Further studies revealed that Hbp1 was a novel target of microRNA-21 and a transcriptional activator of p53. It is well established that p53 is a tumour suppressor and an inhibitor of lipogenesis by inhibiting Srebp1c. As expected, microRNA-21 knockdown led to increased HBP1 and p53 and subsequently reduced lipogenesis and delayed G1/S transition, and the additional treatment of HBP1-siRNA antagonised the effect of microRNA-21-ASO, suggesting that HBP1 mediated the inhibitory effects of microRNA-21-ASO on both hepatic lipid accumulation and hepatocarcinogenesis. Mechanistically, microRNA-21 knockdown induced p53 transcription, which subsequently reduced expression of genes controlling lipogenesis and cell cycle transition. In contrast, the opposite result was observed with overexpression of microRNA-21, which prevented p53 transcription. Conclusions Our findings reveal a novel mechanism by which microRNA-21, in part, promotes hepatic lipid accumulation and cancer progression by interacting with the Hbp1-p53-Srebp1c pathway and suggest the potential therapeutic value of microRNA-21-ASO for both disorders.

MicroRNA-21 (miR-21) is thought to be an oncomir because it promotes cancer cell proliferation, migration, and survival. miR-21 is also expressed in normal cells, but its physiological role is poorly understood. Recently, it has been found that miR-21 expression is rapidly induced in rodent hepatocytes during liver regeneration after two-thirds partial hepatectomy (2/3 PH). Here, we investigated the function of miR-21 in regenerating mouse hepatocytes by inhibiting it with an antisense oligonucleotide. To maintain normal hepatocyte viability and function, we antagonized the miR-21 surge induced by 2/3 PH while preserving baseline expression. We found that knockdown of miR-21 impaired progression of hepatocytes into S phase of the cell cycle, mainly through a decrease in levels of cyclin D1 protein, but not Ccnd1 mRNA. Mechanistically, we discovered that increased miR-21 expression facilitated cyclin D1 translation in the early phase of liver regeneration by relieving Akt1/mTOR complex 1 signaling (and thus eIF-4F-mediated translation initiation) from suppression by Rhob. Our findings reveal that miR-21 enables rapid hepatocyte proliferation during liver regeneration by accelerating cyclin D1 translation.

The incidence of nonalcoholic fatty liver disease (NAFLD) and hyperlipidemia, with their associated risks of endstage liver and cardiovascular diseases, is increasing rapidly due to the prevalence of obesity. Although the mechanisms of NAFLD have been studied extensively, the underlying pathogenesis and the role of microRNAs in this process remain relatively unclear. MicroRNA (miRNA)-dependent posttranscriptional gene silencing is now recognized as a key element of lipid metabolism. Here we report that the expression of microRNA-24 (miR-24) is significantly increased in the livers of high-fat diet-treated mice and in isolated human hepatocytes incubated with fatty acid. Knockdown of miR-24 in those mice caused impaired hepatic lipid accumulation and reduced plasma triglycerides. Bioinformatic and in vitro and in vivo studies led us to identify insulin-induced gene 1 (Insig1), an inhibitor of lipogenesis, as a novel target of miR-24. Inhibition of endogenous miR-24 expression by way of miR-24 inhibitors led to up-regulation of Insig1, and subsequently decreased hepatic lipid accumulation. It is well established that liver-specific deletion of Insig1 leads to higher hepatic and plasma triglyceride levels by inhibiting the processing of sterol regulatory element-binding proteins (SREBPs), transcription factors that activate lipid synthesis. As expected, miR-24 knockdown prevented SREBP processing, and subsequent expression of lipogenic genes. In contrast, the opposite result was observed with overexpression of miR-24, which enhanced SREBP processing. Thus, our study defines a potentially critical role for deregulated expression of miR-24 in the development of fatty liver by way of targeting of Insig1. Conclusion Our findings show a novel mechanism by which miR-24 promotes hepatic lipid accumulation and hyperlipidemia by repressing Insig1, and suggest the use of miR-24 inhibitor as a potential therapeutic agent for NAFLD and/or atherosclerosis.