Cytochrome P450 (P450) enzymes are responsible for biotransformation of xenobiotics, including environmental toxicants and drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcome in either therapeutic efficacy or adverse effects. Nuclear receptors are a class of transcription factors that can regulate expression of P450s at both basal and drug‐induced levels. Some long non‐coding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors in either promotion or repression manner. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug‐induced expression of P450s in liver cells. Four nuclear receptors, hepatocyte nuclear factor 1a (HNF1a), hepatocyte nuclear factor 4a (HNF4a), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) as well as two lncRNAs, HNF1a‐antisense 1 (HNF1a‐AS1) and HNF4a‐antisense 1 (HNF4a‐AS1) were examined in control of basal and drug‐induced expression of 8 different P450 enzymes. Small interfering RNAs (siRNAs) were applied to knock down the nuclear receptors and lncRNAs in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of the nuclear receptors, lncRNAs, and P450s was measured by RT‐PCR and Western blots. Knocking down of HNF1a or HNF4a affected basal expression of numerous P450s as well as CAR and PXR, and also impaired phenobarbital or rifampicin induced expression of several P450s, which may be due to decreased CAR/PXR expression. Moreover, HNF4a knockdown was able to decrease HNF1a expression and showed more potent effect than HNF1a knockdown. Knocking down of CAR or PXR only affected basal and induced expression of their target P450s without affecting expression of HNF1a or HNF4a. These data suggested the existence of a regulatory network in controlling P450 expression in an order through HNF4a to HNF1a to CAP/PXR. Expression of HNF1a‐AS1 and HNF4a‐AS1 is largely dependent on expression of their nearby coding gene HNF1a and HNF4a. Moreover, HNF4a knockdown depleted expression of both lncRNAs, but HNF1a knockdown only depleted HNF1a‐AS1 expression. Knocking down of HNF1a‐AS1 showed a similar regulation trend on the P450s compared to HNF1a or HNF4a knockdown, indicating involvement of this lncRNA in the regulation of P450s. However, knocking down of HNF4a‐AS1 showed an opposite regulation trend compared to HNF1a or HNF4a knockdown, indicating that HNF4a‐AS1 may play an inhibitory role in the HNF4a function. Altogether, out study concludes that there is a transcription regulatory network among the different nuclear receptors and lncRNAs in the regulation of both basal and drug‐induced expression of P450s in liver cells. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day −2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.
The expression of phase-I drug metabolizing enzymes in liver changes dramatically during postnatal liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in liver. However, the role of FXR in regulating ontogeny of phase-I drug metabolizing genes is not clear. Hence, we applied RNA-sequencing to quantify the developmental expression of phase-I genes in both Fxr-null and control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and Fxr-null mice at 6 different ages from prenatal to adult were used. The Fxr-null showed an overall effect to diminish the “day-1 surge” of phase-I gene expression, including cytochrome P450s at neonatal ages. Among the 185 phase-I genes from 12 different families, 136 were expressed, and differential expression during development occurred in genes from all 12 phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The data also suggested new phase-I genes potentially targeted by FXR. These results revealed an important role of FXR in regulation of ontogeny of phase-I genes.
H19 RNA is highly expressed at early postnatal ages and precipitously decreases at a specific time corresponding with increases in expression of genes important for mature liver function, such as drug metabolizing enzymes. H19’s role in the regulation of liver maturation is currently unknown. Using an H19 knockout mouse model to determine the role of H19 in liver development, we quantified gene expression for insulin growth factor signaling, Wnt signaling, key cytochrome P450 (P450) enzymes known to change as the liver develops, and fetal and adult plasma protein produced in liver. In mice lacking H19 expression, liver weights were significantly increased immediately after birth and significant increases were found in the number of actively proliferating cells. Increases in cell proliferation may be due to increases in β-catenin protein affecting Wnt signaling, increases in insulin-like growth factor 2 (IGF2) expression, and/or increases in insulin-like growth factor 1 receptor (IGF1R) expression at the protein level. Loss of targeted inhibition of IGF1R by microRNA 675 (miR-675) may be the cause of IGF1R increases, as miR-675 expression is also abrogated with loss of H19 expression in our model. P450 expression patterns were largely unchanged. No change in the production of plasma proteins was found, indicating H19 may not be important for liver maturation despite its role in controlling cell proliferation during liver growth. H19 may be important for normal liver development, and understanding how the liver matures will assist in predicting drug efficacy and toxicity in pediatric populations.
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