In Silico and In Vitro Identification of MicroRNAs That Regulate Hepatic Nuclear Factor 4α Expression

Ramamoorthy, Anuradha; Li, Lang; Gaedigk, Andrea; Bradford, L. DiAnne; Benson, Eric A.; Flockhart, David A.; Skaar, Todd C.

doi:10.1124/dmd.111.040329

Cited by 63 publications

(68 citation statements)

References 39 publications

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“…In contrast, a clinical study in Han Chinese did not show an association of miR-148a expression with CYP3A4 protein expression (Wei et al, 2013). Multiple different miRNAs target hepatic nuclear factor 4a (HNF4A), a master regulator of many drug-disposition genes that may also be a key contributor to P450 gene expression (Takagi et al, 2010;Ramamoorthy et al, 2012;Wang and Burke, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

et al. 2013

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Rifampin causes drug interactions by altering hepatic drug metabolism. Because microRNAs (miRNAs) have been shown to regulate genes involved in drug metabolism, we determined the effect of rifampin on the expression of hepatic miRNAs. Primary human hepatocytes from seven subjects were treated with rifampin, and the expression of miRNA and cytochrome P450 (P450) mRNAs was measured by TaqMan assays and RNA-seq, respectively. Rifampin induced the expression of 10 clinically important and 13 additional P450 genes and repressed the expression of 9 other P450 genes (P < 0.05). Rifampin induced the expression of 33 miRNAs and repressed the expression of 35 miRNAs (P < 0.05). Several of these changes were highly negatively correlated with the rifampin-induced changes in the expression of their predicted target P450 mRNAs, supporting the possibility of miRNA-induced regulation of P450 mRNA expression. In addition, several other miRNA changes were positively correlated with the changes in P450 mRNA expression, suggesting similar regulatory mechanisms. Despite the interindividual variability in the rifampin effects on miRNA expression, principal components analysis clearly separated the rifampin-treated samples from the controls. In conclusion, rifampin treatment alters miRNA expression patterns in human hepatocytes, and some of the changes were correlated with the rifampin-induced changes in expression of the P450 mRNAs they are predicted to target.

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Section: Introductionmentioning

confidence: 99%

“…For example, there are at least 38 genetic polymorphisms in the P450 genes that are predicted to either create or destroy miRNA binding sites (Ramamoorthy et al, 2012). A single-nucleotide polymorphism in the miR-34a binding site of the HNF4A gene (rs11574744) alters the targeting of that gene by miR-34a (Ramamoorthy et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

et al. 2013

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“…HNF-4α is required for the development of the liver and it is known as a "master regulator" that regulates the expression of many genes. 16) HNF-4α has been reported to modulate CYP expression in the liver by interacting with xenosensor receptors (e.g. PXR and CAR), glucocorticoid receptors, feeding-fasting cycle target PGC-1α, sexual-dimorphism factor Stat5b, and other liver-enriched factors, such as C/EBPs.…”

Section: Discussionmentioning

confidence: 99%

Human Hepatocarcinoma Functional Liver Cell-4 Cell Line Exhibits High Expression of Drug-Metabolizing Enzymes in Three-Dimensional Culture

Kato

Shigemoto

Akiyama

et al. 2014

Biological & Pharmaceutical Bulletin

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The expression levels of CYP and uridine diphosphate-glucuronosyl transferase (UGT) are lower in hepatocellular carcinoma cell lines than in human primary hepatocytes. However, a functional liver cell (FLC)-4 cell line that has a greater capacity to secrete liver-specific proteins than other hepatocellular carcinoma cells has recently been established. A three-dimensional culture using Engelbreth-Holm-Swan (EHS) gel induces the secretion of liver-specific proteins via the induction of hepatocyte nuclear factor-4α (HNF-4α). The aim of this study was to evaluate the mRNA expression of the enzymes CYP and UGT in FLC-4 and HepG2 cells in monolayer and three-dimensional cultures using EHS gel. The mRNA levels of HNF-4α, albumin, pregnane X receptor (PXR), constitutive androstane receptor (CAR), CYPs (1A2, 2E1, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) were determined using real-time reverse transcription (RT) PCR. In a monolayer culture, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, and 1A9) were higher in FLC-4 cells than in HepG2 cells. In FLC-4 cells, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C8, 2C19, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) significantly increased in three-dimensional culture. FLC-4 cells cultured in EHS gel showed significantly increased expression levels of CYPs and UGTs. The results of this study suggest that human hepatocellular carcinoma FLC-4 cells cultured in EHS gel show potential for use in studying in vitro drug metabolism.Key words CYP; uridine diphosphate-glucuronosyl transferase; expression; functional liver cell; threedimensional culture Human hepatocellular carcinoma cell lines (e.g. HepG2, Hep3B, and HuH7) are widely used to study drug metabolism and toxicology in vitro. The expression levels of CYP and uridine diphosphate-glucuronosyl transferase (UGT) in hepatocellular carcinoma cell lines are known to be lower than those in human primary hepatocytes. 1) However, under typical culture conditions, human primary hepatocytes are unable to sustain stable hepatic functions, such as production of drugmetabolizing enzymes.2) Therefore, hepatic microsomes are usually used in in vitro experiments related to drug metabolism. Functional liver cell (FLC) lines have been established from resected liver tumors of Japanese patients. These cells synthesized and secreted human albumin, alpha-fetoprotein, and other proteins in vitro. 3)Many reports have shown that the differentiation status of primary culture hepatocytes could be enhanced by threedimensional culture.4,5) Engelbreth-Holm-Swan (EHS) gel, derived from mouse EHS sarcoma, induces the expression of hepatocyte nuclear factor-4α (HNF-4α).6) HNF-4α has been reported to play a significant role in the regulation of many genes expressed in the liver and to trans-activate the human pregnane X receptor (PXR) gene. 7,8) Human PXR is known to be a transcriptional regulator of genes encoding drug-metabolizing enzymes, including C...

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“…Rifampin-induced miRNA expression changes are correlated with rifampin treatment changes in PXR and P450 [6,[10][11][12]. MiRNA can indirectly influence rifampin-induced drug deposition through gene regulation [11,[13][14][15]. Proteins are synthesized from mRNA templates through a highly conserved process, many studies have reported that the functions of miRNAs can be understood by analyzing miRNAs-regulated mRNA-related PIN [16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Integrative network analysis of rifampin-regulated miRNAs and their functions in human hepatocytes

Wang

et al. 2015

BME

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Abstract.Rifampin is an important drug used in the treatment of tuberculosis, and it increases the drug metabolism in human hepatocytes. Previous studies have shown that rifampin can indirectly influence drug deposition through the regulation of molecular interactions of miRNA, PXR and other genes. The potential functions of miRNAs associated with rifampininduced drug disposition are poorly understood. In this study, significantly differentially expressed miRNAs (SDEM) were extracted and used to predict the miRNA-regulated co-expression target genes (MCeTG). Additionally, a miRNA-regulated co-expressed protein interaction network (MCePIN) was constructed for SDEM by extending from the protein interaction network (PIN). The functioning of the miRNAs were analyzed using GO analysis and KEGG pathway enrichment analysis. A total of 20 miRNAs belonging to SDEM were identified, and 632 miRNA-regulated genes were predicted. The MCePIN was constructed by extending from PIN, and 10 miRNAs and 33 genes that are relevant to 7 functions, including response to wounding, wound healing, response to drug, defense response, inflammatory response, liver development and drug metabolism, were discerned. The results provided by this study offer valuable insights into the effect of rifampin on miRNAs, genes and protein levels.

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In Silico and In Vitro Identification of MicroRNAs That Regulate Hepatic Nuclear Factor 4α Expression

Cited by 63 publications

References 39 publications

Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

Human Hepatocarcinoma Functional Liver Cell-4 Cell Line Exhibits High Expression of Drug-Metabolizing Enzymes in Three-Dimensional Culture

Integrative network analysis of rifampin-regulated miRNAs and their functions in human hepatocytes

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