Hepatocyte nuclear factor (HNF) 4␣ is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4␣. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3-untranslated region (3-UTR), and those for miR-34a (MRE34a) were identified in the 3-UTR in HNF4␣ mRNA. The HNF4␣ protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4␣ mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3-UTR of HNF4␣ was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4␣ coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4␣ by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4␣ was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.Human hepatocyte nuclear factor 4␣ (HNF4␣, NR2A1), 3 which belongs to the nuclear hormone receptor superfamily, is highly expressed in liver and regulates the expression of various genes involved in the synthesis/metabolism of fatty acid, cholesterol, glucose, and urea (1). It is well recognized that endo/xenobiotic-metabolizing enzymes such as cytochrome P450s (CYPs), UDP-glucuronosyltransferases, sulfotransferase as well as ATP-binding cassette transporters, organic anion transporters and organic cation transporters are under the control of HNF4␣ (2). HNF4␣ transactivates the expression of target genes not only via direct binding to their regulatory sequences but also through the regulation of other transcriptional factors such as pregnane X receptor and constitutive androstane receptor, which regulate these target genes. HNF4␣ forms large transcriptional regulatory networks in the liver. Therefore, it is believed that the change of HNF4␣ expression has a great impact upon the function of the liver.Bile acids are important regulatory molecules mediating cholesterol synthesis and glucose metabolism as well as their own synthesis (3). It is well known that HNF4␣ positively regulates the expression of bile acid-synthesizing enzymes such as CYP7A1 and CYP8B1. When bile acids are accumulated, the HNF4␣-mediated transactivation is inhibited by short heterodimer p...

MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121–317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6–35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.

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The profiling of the selected miRNAs can be useful to distinguish different types of liver diseases.

SDS software v.2.4. The expression levels were evaluated using the comparative cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Statistical analysisThe data are expressed as the means ± SD. The comparisons of two groups were made using Mann-Whitney's U-test. A value of P < 0.05 was considered statistically significant. Results ALT, AST, and histopathology of the liverAfter the administration of APAP and MP, the plasma ALT and AST levels were significantly elevated (Fig. 1A). The histopathological analysis revealed that APAP caused hepatocellular necrosis at the pericentral regions, whereas MP caused hepatocellular necrosis at the periportal region (Fig. 1B). These two models were used for the subsequent studies. miRNA expression at the pericentral and periportal regions of the liverThe miRNA expression at pericentral and periportal regions of the liver was determined by TaqMan microRNA array analysis. The numbers of the detected miRNAs and the miRNAs whose expression exceeded the cutoff (Ct < 32) are shown in Table 1. No large difference was observed in these numbers between the pericentral and periportal regions within a group or between the treated and control groups. Among the miRNAs with Ct values less than 32 in all of the groups, 125 miRNAs were common to all of the groups; therefore, these 125 miRNAs were used for the subsequent analyses. Fig. 2 shows the heat maps of the expression of the 125 miRNAs in each sample; note that the miRNAs are ordered based on descending expression level in the pericentral region of the control rats. The top three miRNAs that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 exhibited high expression in both the pericentral and periportal regions in all of the samples are miR-709, miR-122, and miR-720. The comparison of the miRNA levels between the control rats and fasted-control rats revealed that 36 miRNAs presented higher levels and eight miRNAs exhibited lower levels at the pericentral region of the fasted-control rats (Tables 1 and 2). At the periportal region, 52 miRNAs and two miRNAs presented higher and lower levels, respectively, in the fasted-control rats compared with the control rats. Among these miRNAs, 17 miRNAs, which are shown in bold, were found in both the pericentral and periportal regions. Thus, the results demonstrate that fasting affects the expression of some miRNAs in the liver. The comparison of the miRNA levels between the pericentral and periportal regions in control rats showed that 27 miRNAs exhibited higher expression levels in the pericentral region and that 22 miRNAs presented higher expression levels in the periportal region compared with t...