Miki Nakajima

MicroRNA regulates human vitamin D receptor

Most of the biological effects of 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3) are elicited by the binding to vitamin D receptor (VDR), which regulates gene expression. Earlier studies reported no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through translational repression or mRNA degradation. A potential miR‐125b recognition element (MRE125b) was identified in the 3′‐untranslated region of human VDR mRNA. We investigated whether VDR is regulated by miR‐125b. In luciferase assays using a plasmid containing the MRE125b, the antisense oligonucleotide for miR‐125b significantly increased (130% of control) the reporter activity in KGN cells, whereas the precursor for miR‐125b significantly decreased (40% of control) the reporter activity in MCF‐7 cells, suggesting that miR‐125b functionally recognized the MRE125b. By electrophoretic mobility shift assays, it was demonstrated that the overexpression of miR‐125b significantly decreased the endogenous VDR protein level in MCF‐7 cells to 40% of control. 1,25(OH)2D3 drastically induced the CYP24 mRNA level in MCF‐7 cells, but the induction was markedly attenuated by the overexpression of miR‐125b. In addition, the antiproliferative effects of 1,25(OH)2D3 in MCF‐7 cells were significantly abolished by the overexpression of miR‐125b. These results suggest that the endogenous VDR level was repressed by miR‐125b. In conclusion, we found that miR‐125b posttranscriptionally regulated human VDR. Since the miR‐125b level is known to be downregulated in cancer, such a decrease may result in the upregulation of VDR in cancer and augmentation of the antitumor effects of 1,25(OH)2D3.

Human CYP2E1 is regulated by miR-378

Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.

MicroRNAs Regulate Human Hepatocyte Nuclear Factor 4α, Modulating the Expression of Metabolic Enzymes and Cell Cycle

Hepatocyte nuclear factor (HNF) 4α is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4α. 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 HNF4α mRNA. The HNF4α protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4α 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 HNF4α was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4α 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 HNF4α 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 HNF4α 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 CYP24 Catalyzing the Inactivation of Calcitriol Is Post-Transcriptionally Regulated by miR-125b

Human vitamin D3 hydroxylase (CYP24) catalyzes the inactivation of 1α,25-dihydroxyvitamin D3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3′-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P < 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.

PPARα Is Regulated by miR-21 and miR-27b in Human Liver

ABSTRACTPurposePeroxisome proliferator-activated receptor α (PPARα) is an important transcriptional factor that regulates genes encoding endo/xenobiotic enzymes and lipid metabolizing enzymes. In this study, we investigated whether microRNAs (miRNAs) are involved in the regulation of PPARα in human liver.MethodsPrecursor or antisense oligonucleotide for miR-21 or miR-27b was transfected into HuH7 cells; expression of PPARα and acyl-CoA synthetase M2B was determined by Western blot and real-time RT-PCR. Luciferase assay was performed to identify the functional miRNA recognition element (MRE). Expression levels of PPARα, miR-21, and miR-27b in a panel of 24 human livers were determined.ResultsThe overexpression and inhibition of miR-21 or miR-27b in HuH7 cells significantly decreased and increased the PPARα protein level, respectively, but not PPARα mRNA level. The miRNA-dependent regulation of PPARα affected the expression of its downstream gene. Luciferase assay identified a functional MRE for miR-21 in the 3′-untranslated region of PPARα. In human livers, the PPARα protein levels were not correlated with PPARα mRNA, but inversely correlated with the miR-21 levels, suggesting a substantial impact of miR-21, although the contribution of miR-27b could not be ruled out.ConclusionsWe found that PPARα in human liver is regulated by miRNAs.

Toxicological Implications of Modulation of Gene Expression by MicroRNAs

MicroRNAs (miRNAs) are a large family of non-coding RNAs that are evolutionarily conserved, endogenous, and 21-23 nucleotides in length. miRNAs regulate gene expression by targeting messenger RNAs (mRNAs) by binding to complementary regions of transcripts to repress their translation or mRNA degradation. miRNAs are encoded by the genome, and more than 1000 human miRNAs have been identified so far. miRNAs are predicted to target ∼60% of human mRNAs and are expressed in all animal cells and have fundamental roles in cellular responses to xenobiotic stresses, which affect a large range of physiological processes such as development, immune responses, metabolism, tumor formation as well as toxicological outcomes. Recently, many reports concerning miRNAs related to cancer have been published; however, the miRNA research in the metabolism of xenobiotics and endobiotics and in toxicology has only recently been established. This review describes the current knowledge on the miRNA-dependent regulation of drug-metabolizing enzymes and nuclear receptors and its potential toxicological implications. In this review, miRNAs with reference to target prediction, potential modulation of toxicology-related changes of miRNA expression, role of miRNA in immune-mediated drug-induced liver injury, miRNA in plasma as potential toxicological biomarkers, and relevance of miRNA-related genetic polymorphisms are discussed.

Plasma MicroRNA Profiles in Rat Models of Hepatocellular Injury, Cholestasis, and Steatosis

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.

MicroRNAs from biology to future pharmacotherapy: Regulation of cytochrome P450s and nuclear receptors

MicroRNAs (miRNAs) are a family of short, non-coding RNAs whose final product is a 22-nucleotide functional RNA molecule. They regulate the expression of target genes by binding to complementary regions of transcripts to repress their translation or promote mRNA degradation. Since miRNAs regulate every aspect of cellular function, their dysregulation is associated with a variety of diseases including cancer, diabetes, and cardiovascular diseases. Therefore, miRNAs are now considered new therapeutic targets. However, the roles of miRNAs in the metabolism of xenobiotics and endobiotics have only recently been revealed. This review describes the current knowledge on the regulation of cytochrome P450s and nuclear receptors by miRNAs, the physiological and clinical significance. The miRNA expression is readily altered by chemicals, carcinogens, drugs, hormones, stress, or diseases, and the dysregulation of specific miRNAs might lead to changes in the drug metabolism potency or pharmacokinetics as well as pathophysiological changes. In the field of pharmacogenomics, the evaluation of miRNA-related polymorphisms would provide useful information for personalized medicine. Utilizing miRNAs opens a new era in the fields of drug metabolism and pharmacokinetics as well as toxicology.

Halothane-Induced Liver Injury is Mediated by Interleukin-17 in Mice

Drug-induced liver injury is a major problem in drug development and clinical drug therapy. In most cases the mechanisms are still unknown, thus, it is difficult to predict or prevent these reactions. It has been known that halothane, an inhaled anesthetic, induces liver injury. To investigate the mechanisms of halothane-induced liver injury, we used a recently established mouse model of liver injury. The expression of transcription factors and cytokines specific for Th1 and Th2 (helper T cells), respectively, were compared between BALB/c and C57BL/6 mice. The mRNA expression ratios of mouse T-bet(a Th1-specific transcription factor)/GATA-binding protein (GATA-3, a Th2-specific transcription factor) and interferon gamma/interleukin (IL)-10 were lower in BALB/c mice compared with C57BL/6 mice, suggesting that a typical Th1 or Th2-dominant response could not be distinguished in halothane-induced liver injury. We observed increases of the plasma IL-17 level and hepatic macrophage inflammatory protein 2 expression in halothane-administrated BALB/c mice, as well as neutrophil infiltration. Neutralization of IL-17 suppressed the hepatotoxic effect of halothane. Administration of recombinant IL-17 (1 microg per mouse, single ip) to the halothane-treated mice resulted in a remarkable increase of alanine and aspartate aminotransferases. In conclusion, we demonstrated that IL-17 is involved in the halothane-induced liver injury.

Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation

Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6β-hydroxylase activity (670 pmol · min−1 · mg−1). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the drug-induced cytotoxicity associated with CYP3A4.