Fumonisin B1 modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b
Introduction
Fusarium verticilloides, a ubiquitous soil fungus and common contaminant of corn worldwide, produces the carcinogen fumonisin B1 (FB1) that is responsible for a wide range of species-specific toxicoses (Norred et al., 1998). FB1, a structural analogue of sphingoid bases, is implicated in cancer promotion as it disrupts sphingolipid, phospholipid and fatty acid metabolism, which play a major role in the modulation of apoptosis and cell proliferation pathways (Gelderblom and Marasas, 2012). An elevation of free sphingoid bases by FB1 can induce apoptosis, whereas production of sphingosine-1-phosphate or inhibitors of ceramide biosynthesis can also inhibit apoptosis (Norred et al., 1998). Chronic feeding of animals with FB1 caused liver cancer in mice and rats (Howard et al., 2001). At present little is known about FB1 genotoxicity however, it is known that FB1 induces necrosis as well as apoptosis in the liver (Gelderblom and Marasas, 2012).
MicroRNAs (miRNA) are small non-coding RNAs regulating the expression of genes involved in various biological processes including cell proliferation and apoptosis (Ambros, 2004). Mature functional miRNAs (approximately 22 nucleotides in length) generated from long primary miRNA transcripts, control gene expression at the post-transcriptional level by either degrading or repressing target mRNAs. MiRNAs are thus selected gene regulatory molecules with each cell type likely to have a specific miRNA milieu to control gene expression (Chen, 2005). The functional miRNAs have been predicted to regulate expression of approximately 30% of all human genes (Lewis et al., 2005). MiRNAs play an important role in coordinating many cellular processes such as regulating apoptosis, proliferation, differentiation, development and metabolism (Gusev, 2008, Maziere and Enright, 2007).
Some miRNAs expressed in cancer were found to regulate the expression of signalling molecules such as cytokines, growth factors, transcription factors and both pro- and anti-apoptotic genes; whilst some miRNAs may function as tumour suppressors or conversely oncogenes in cells (Calin and Croce, 2006). MiRNAs may therefore be one of the key regulators of tumorigenesis.
Tsuchiya et al. (2006) found that cytochrome P450 (CYP1B1), a superfamily of drug metabolising enzymes, was a target of miR-27b (Tsuchiya et al., 2006). CYP1B1, a highly expressed enzyme in oestrogen target tissues, catalyzes the activation of various procarcinogens and promutagens, polycyclic aromatic hydrocarbons and aryl amines (Shimada et al., 1996) and 17β-estradiol (Lee et al., 2003). In addition CYP1B1 is highly expressed in cancerous tissues (e.g. breast cancer) and a near-perfect matching sequence was identified with miR-27b in the 3′-untranslated region (UTR) of CYP1B1 (Tsuchiya et al., 2006). It was concluded that CYP1B1 was post-transcriptionally regulated by miR-27b and that the expression level of miR-27b was decreased accompanied by high levels of CYP1B1 in the cancerous liver tissue (Tsuchiya et al., 2006).
In response to noxious stimulation by FB1, and as a result of aberrant gene expression, cells may alter their normal miRNA profiles and create a micro-environment which facilitates carcinogenesis. This toxin is a potent liver carcinogen, however there is little consensus on the precise mechanism of neoplastic transformation. The effect of FB1 on miRNA has not been previously investigated and it is likely that cells may change their miRNA milieu in response to FB1. In this study we screened for changes in miRNA expression profiles in human hepatoma cells (HepG2) following exposure to FB1. Interestingly, we show that FB1 induced significant changes in miR-27b expression and in turn modulates CYP1B1 expression. We suggest that FB1- induced modulation of miR-27b in hepatic cells is an additional mode of hepatic neoplastic transformation.
Section snippets
Treatment
Approximately 1.5 × 106 HepG2 cells were plated in sterile 25 cm3 flasks in complete culture media [Eagle's minimum essential medium, 10% foetal calf serum, 1% l-glutamine and 1% penstrep-fungizone] and incubated overnight at 37 °C in a humidified incubator with a supply of 5% CO2. A stock solution of 5 mM FB1 was prepared in 0.1 M phosphate buffered saline (PBS). HepG2 cells were treated with a range (0–1000 μM) of concentrations of FB1. A dose dependent decline in HepG2 cell viability was observed
MicroRNA profiles in fumonisin B1 treated HepG2 cells
MiRNA profiling showed that FB1 induced differential regulation of miRNAs in HepG2 cells. Expression profile heatmap shows robust signals from control and treated cells (Fig. 1A). This indicated good data integrity and that dataset is suitable to mine for candidate miRNAs that may be differentially regulated by FB1 treatment. Each point in the expression profile plot (Fig. 1B) represents a single miRNA species with total coverage of 84 unique miRNAs. This analysis shows that miRNA expression
Discussion
The current mechanism of action associated with FB1 is inhibition of ceramide synthase and the disruption of sphingolipid biosynthesis. The sphingolipid dysregulation by FB1 strongly correlates with mammalian pathogenesis, including cancer. However, disruption of sphingolipid metabolism alone may not fully explain the proliferative and ultimately oncogenic properties of FB1. In this report, we present evidence that FB1 significantly down-regulates miR-27b and concomitantly up-regulates both
Conflicts of interest
The authors declare that there are no conflicts of interest.
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References (22)
- et al.
miR-27b controls venous specification and tip cell fate
Blood
(2012) - et al.
Differential expression of CYP1A1, CYP1A2, CYP1B1 in human kidney tumours
Cancer Letters
(1999) - et al.
Fumonisin B1 induces global DNA hypomethylation in HepG2 cells – an alternative mechanism of action
Toxicology
(2014) Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA
Methods
(2008)- et al.
Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets
Cell
(2005) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001) - et al.
Prediction of microRNA targets
Drug Discovery Today
(2007) - et al.
CYP1B1 expression in prostate is higher in the peripheral than in the transition zone
Cancer Letters
(2004) The functions of animal microRNAs
Nature
(2004)- et al.
Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression
Molecular Pharmacology
(2005)
MicroRNA signatures in human cancers
Nature Reviews. Cancer
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Present address: Department of Microbiology and Immunobiology, Harvard Medical School 77, Avenue Louis Pasteur, Boston, MA 02115 NRB – 1052, United States.