Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
A dual inhibition: microRNA-552 suppresses both transcription and translation of cytochrome P450 2E1
Introduction
MicroRNAs play important regulatory roles in a vast range of biological processes including development and diseases [1], [2]. They direct post-transcriptional gene silencing (PTGS) through limited base-pairing in their seed region and the complementary sequences in the 3′-untranslated region (UTR) of an mRNA. The PTGS depends on paring of the seed region (usually residues 2–8) of a miRNA, and pairing outside this 7 nucleotide (nt) core site is thought to provide means of conferring added specificity [3]. Biogenesis of most miRNAs originates from large primary transcripts (pri-miRNAs) and following cleavage and nucleocytoplasmic export, mature miRNAs are processed in cytoplasm to downregulate target genes by either mRNA cleavage or translation repression [4].
It is generally accepted that miRNAs function in the cytoplasm, however, mature miRNAs have also been found in the nucleus [5], [6], [7], [8], with abundant putative target sites in gene promoter [9], [10]. It has been shown that dsRNAs with sequence complementarity to promoter regions induce transcriptional gene silencing (TGS) in mammalian cells through heterochromatin assembly [11], [12]. Although the function of nuclear miRNA is not entirely known, emerging findings supported the possibility of miRNA-induced TGS [10], [13], [14], [15], [16], [17], [18]. A study with exogenous miRNA mimics reported that miR-423-5p suppressed progesterone receptor (PR) gene transcription by recruiting Ago2 to an antisense non-coding RNA transcribed from the PR promoter, decreasing RNA polymerase II occupancy and increasing histone H3 lysine dimethylation (H3K9me2) at the PR promoter [13]. MiR-320 which was transcribed from the promoter of the POLR3D gene suppressed PLOR3D transcription in cis through directing epigenetic changes [10]. Multiple other miRNAs have since been reported targeting specific promoters [16], inducing epigenetic silencing and functioning in the regulation of cellular senescence [14], granulopoiesis [15] and nerve regeneration [17] via targeting multiple genes.
Although miRNAs have been found localized in both cytoplasm and nucleus [5], [6], [7], [8], whether miRNA in both subcellular locations could function is not entirely known. Sequence complementarity of miRNAs to the targets is important for both PTGS and TGS function of miRNAs. It is possible that a miRNA negatively regulates its targets at both post-transcriptional and transcriptional levels. Here, in search for miRNAs to affect levels of CYP2E1, a CYP isoform responsible for the oxidation of chemicals such as ethanol, arachidonic acid or fatty acids, which is also an effective generator of reactive oxygen species and involved in hepatic insulin resistance [19], [20], [21], [22], [23], we accidentally found that hsa-miR-552 localizes to both cytosol and nucleus and regulates CYP2E1 expression at both PTGS and TGS levels.
Section snippets
MiR-552 inhibits CYP2E1 expression in human hepatoma cells
We initially explored the miRNAs that can affect the levels of CYP2E1, whose expression levels were elevated in a variety of pathophysiological conditions such as alcoholic or non-alcoholic fatty liver diseases [24], [25]. Prediction using Targetscan [26], MicroCosm Targets [27] and microRNA.org databases [28] identified 31 miRNAs that could potentially bind to the 3′-UTR of human CYP2E1. Analysis of its promoter regions from − 1 to − 1500 bp upstream of the transcription start site via miRBase
Discussion
Here, we report that miR-552 is a novel regulator of expression of CYP2E1 known as a vital player in the metabolism of ethanol and other low molecular weight chemicals [37]. Since expression of CYP2E1 with inter-individual variation is altered in multiple pathophysiological conditions such as alcoholic liver diseases [25], [38], [39], [40], [41], [42], its expression is induced by the chemical substrates through decreased protein degradation [43], [44], and insulin regulates the miRNAs which
Animal experiments
The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Neuroscience, Shanghai, China. Total liver proteins were extracted from C57/BL6 mice.
Cell culture, transfection and treatment
The human cell lines PLC/PRF/5 and HepG2 were purchased from American Type Culture Collection (ATCC), others from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen), supplemented with 10% fetal
Funding
This work was supported by grants from the National Natural Science Foundation of China [81,130,081] and the National Science and Technology Major Project [2012ZX09302-003, 2015ZX09102005].
Transparency document
Acknowledgement
We thank LiHua Wang for UV melting curve analysis, ChaoLan Huang and Ping Wu for mass spectrometry, Ziran Zhao for data analysis and presentation.
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Prenatal ethanol exposure induces dynamic changes of expression and activity of hepatic cytochrome P450 isoforms in male rat offspring
2022, Reproductive ToxicologyCitation Excerpt :UBCT/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were activated in the liver under alcohol exposure[51], thus, we speculated that ubiquitination might participate in the post-translational modification of CYPs and reduce the protein content. In addition, post-transcriptional modifications also include miRNA-mediated mRNA degradation, and a series of miRNAs are responsible for CYP2E1 downregulation by binding to certain regions of mRNA, including miRNA-552, miR-132, miR-212 and et al.[52]. Moreover, ethanol-induced intracellular oxidative stress activated Akt and protein kinase C signaling pathway, which consequentially caused CYP2E1 protein phosphorylation and increased its susceptibility to degradation[53,54].
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Present Address: Center for Advanced Therapeutic Strategies for Brain Disorders, Roskamp Institute, Sarasota, FL 34243, USA.