Fumonisin B1 modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b

doi:10.1016/j.toxlet.2014.02.026

Toxicology Letters

Volume 227, Issue 1, 16 May 2014, Pages 50-55

https://doi.org/10.1016/j.toxlet.2014.02.026 Get rights and content

Highlights

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Fumonisin B₁ significantly downregulates miR27b in HepG2 cells.
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FB₁, significantly upregulates CYP1B1 mRNA and protein expression in HepG2 cells.
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CYP1B1 is post-transcriptionally regulated by miR-27b after HepG2 exposure to FB₁.

Abstract

Fumonisin B₁ (FB₁), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in hepatocellular carcinoma promoting activity in humans and animals. MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression via translational repression. Human cytochrome P450 (CYP1B1) is highly expressed in oestrogen target tissues and catalyzes the metabolic activation of many procarcinogens. The aim of our study was to investigate the effect of FB₁ on miR-27b suppression and its effect on CYP1B1 modulation in a human hepatoma cell line (HepG2). MiR27b and CYP1B1 expressions were evaluated in HepG2 cells by quantitative PCR. In order to directly assess the effect of miR-27b on CYP1B1 mRNA levels, cells were transfected with the mimic to miR-27b. CYP1B1 protein expression was measured using Western blot. FB₁ significantly down-regulated (11-fold) expression of miR-27b in HepG2 cells; whilst CYP1B1 mRNA and protein expression was significantly upregulated by 1.8-fold and 2.6-fold, respectively. CYP1B1 is post-transcriptionally regulated by miR-27b after HepG2 exposure to FB₁. FB₁-induced modulation of miR-27b in hepatic cells may be an additional mode of hepatic neoplastic transformation.

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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Fumonisin B1 (FB1) is a contaminant that commonly present in the global environment, especially in food and feed. Epidemiologic studies have shown that esophageal cancer is associated with fumonisin toxicity. However, the molecular mechanism of FB1-induced esophageal cancer is unclear. In this research, the molecular mechanism of FB1-induced cell carcinogenesis in human esophageal epithelial cells line (HEEC) was explored. We found that FB1 (0.3125–5 μM) could promote cell proliferation, and the same phenomenon was found in a 3D cell model. FB1 could also accelerate cell migration. The expression levels of DNA damage markers were significantly increased after FB1 exposure. Meanwhile, the expression levels of cell cycle-regulated proteins and cancer-related genes were abnormal. Furthermore, FB1 significantly upregulated the histone deacetylase (HDAC) expression and activated the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) signalling pathway. The HDAC inhibitor trichostatin A (TSA) could repressed FB1-promoted cell proliferation and abnormal phenomenon induced by FB1. Moreover, myriocin (ISP-1) could relieve FB1-enhanced HDAC expression and cell proliferation, which implied that ISP-1 may be used to block the fumonisin toxicity in the future. Our findings suggested that the HDAC/PI3K/Akt signalling pathway is a novel mechanism for FB1-induced cell carcinogenesis in HEEC and provided new ideas for the prevention and control of fumonisin toxicity, subsequently avoiding adverse effects on the ecosystem and human health.
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¹: Present address: Department of Microbiology and Immunobiology, Harvard Medical School 77, Avenue Louis Pasteur, Boston, MA 02115 NRB – 1052, United States.

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Fumonisin B1 modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b

Highlights

Abstract

Introduction

Section snippets

Treatment

MicroRNA profiles in fumonisin B1 treated HepG2 cells

Discussion

Conflicts of interest

Transparency document

Blood

Cancer Letters

Toxicology

Methods

Cell

Methods

Drug Discovery Today

Cancer Letters

The functions of animal microRNAs

Nature

Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression

Molecular Pharmacology

MicroRNA signatures in human cancers

Nature Reviews. Cancer

Fumonisin B₁ modulates expression of human cytochrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b

MicroRNA profiles in fumonisin B₁ treated HepG2 cells