The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation

https://doi.org/10.1016/j.bbagrm.2012.12.009Get rights and content

Abstract

Hepatocyte nuclear factor (HNF)-4α is a key member of the transcription factor network regulating hepatocyte differentiation and function. Genetic and molecular evidence suggests that expression of HNF-4α is mainly regulated at the transcriptional level. Activation of HNF-4A gene involves the interaction of distinct sets of transcription factors and co-transcription factors within enhancer and promoter regions. Here we study the inhibitory effect of microRNAs (miRNAs) on the 3′-untranslated region (3′-UTR) of HNF-4A mRNA. The potential recognition elements of a set of miRNAs were identified utilizing bioinformatics analysis. The family members of miR-34 and miR-449, including miR-34a, miR-34c-5p and miR-449a, share the same target elements located at two distinct locations within the 3′-UTR of HNF-4A. The over-expression of miR-34a, miR-34c-5p or miR-449a in HepG2 cells led to a significant decrease in the activity of luciferase reporter carrying 3′-UTR of HNF-4A. The repressive effect on reporter activity was partially or fully eliminated when one or two of the binding site(s) for miR-34a/miR-34c-5p/miR-449a were deleted within the 3′-UTR. The protein level of HNF-4α was dramatically reduced by over-expression of miR-34a, miR-34c-5p and miR-449a, which correlates with a decrease in the binding activity of HNF-4α and transactivation of HNF-4α target genes. These results suggest that the recognition sites of miR-34a, miR-34c-5p and miR-449a within 3′-UTR of HNF-4A are functional. The mechanism of down-regulation of the binding activity and transactivation of HNF-4α by the miRNAs involves the decrease in HNF-4α protein level via miRNAs selectively targeting HNF-4A 3′-UTR, leading to the translational repression of HNF-4α expression.

Highlights

► HNF-4 plays a crucial role in the development and maintenance the liver phenotype. ► Specific miRNAs down-regulate HNF-4 expression by targeting 3′-UTR of HNF-4 mRNA. ► The miRNA dependent regulation affects the transactivation of HNF-4 target genes. ► The regulatory mechanism of miRNAs may have important impacts on liver phenotype.

Introduction

The liver-enriched transcription factors are part of a complex transcriptional network which is responsible for both determination and maintenance of the hepatic phenotype. This network is established by a number of auto-regulatory and cross-regulatory pathways securing balance and high-level expression of genes in the hepatocytes [1], [2], [3], [4]. Hepatocyte nuclear factor-4α (HNF-4α) is considered to be a critical factor in the development and maintenance of the hepatic phenotype and to be at the top of the transcription factor cascade. The pivotal role of HNF-4α in the maintenance of the hepatic phenotype is highlighted by the severe metabolic defects in mice where HNF-4α was inactivated in the adult liver [5] and by the exceptionally high number of potential target genes revealed by genome-scale target search studies [6], [7]. In the adult human liver, HNF-4α was found to occupy approximately 12% of the genes represented in a 13 K DNA microarray and approximately 42% of those bound by RNA polymerase II [7]. HNF-4α has been shown to contribute to human disease, such as, maturity onset diabetes of the young (MODY1), which results from haploinsufficiency of the HNF-4A gene [8], [9]. Previous work by our group has demonstrated that HNF-4α plays a role in the liver's response to systemic injury and the development of the hepatic acute phase response phenotype [10], [11]. These studies have established that HNF-4α acts as a key regulator in liver function, which raises the importance of understanding how the activity and expression of HNF-4α are regulated.

The regulation of the HNF-4A gene has been well studied. The HNF-4A gene is regulated mainly at the level of transcription. Two main regulatory regions have been identified: the proximal promoter and a distant enhancer are located upstream of the transcription start site [12], [13], [14]. It has been shown that activation of the HNF-4A gene requires the action of HNF-1α and HNF-6 on the proximal promoter, which communicates via a looping mechanism with a distant enhancer bound by HNF-1α, HNF-3β and C/EBPα [12], [13]. In addition, previous studies have shown that HNF-4α activity is also modified by post-transcriptional mechanisms, such as, phosphorylation [15], [16], [17], acetylation [18], and protein–protein interactions with other factors or co-regulators [19], [20], [21], suggesting that HNF-4α activity can be controlled by multiple pathways.

The functional elements within the 3′-untranslated region (3′-UTR) of various mRNAs have been shown to play an important role in translation and stability of these mRNAs [22] with significant impact on gene regulation and phenotype. However, the potential mechanisms of regulating HNF-4α expression and function by targeting the 3′-UTR of HNF-4A mRNA via microRNAs (miRNAs) remain to be further explored.

MiRNAs are endogenous non-coding RNA molecules that mainly mediate post-transcriptional regulation of gene expression by targeting sequence specific regions in the 3′-UTR of mRNA and repressing their translations into proteins [23], [24]. MiRNAs are the most abundant regulators of gene expression in the human genome, and have enormous regulatory potential. Hundreds of miRNAs have been identified in humans. MiRNAs are predicted to control approximately 30% of the genes within the human genome [25], [26], and participate in the regulation of various biological processes [27]. It has been recently reported that HNF-4α is a gene target of a number of miRNAs [28], [29], [30], [31], which provides a potential regulatory mechanism by which HNF-4α function can be modulated by miRNAs. In the present study, we focus our work to validate and analyze the effect of several members of miRNA-34 and miRNA-449 on HNF-4α expression and the ability of HNF-4α to bind and transactivate its target genes. Our results indicate that miRNAs (miR-34a, miR-34c-5p and miR-449a) not only post-transcriptionally regulate the expression of HNF-4α, but also affect HNF-4α binding and transactivation of its target genes by a mechanism of selectively targeting of the HNF-4A 3′-UTR.

Section snippets

Cell culture

The human hepatoma cell line, HepG2, was obtained from ATCC (HB-8065), and maintained in Dulbecco's modified Eagle's medium, supplemented with 100 units/ml of penicillin, 100 μg/ml of streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (Mediatech, Herndon, VA) at 37 °C in a humidified atmosphere with 5% CO2.

The miRNA target prediction

The miRNA target predictions were performed using TargetScan version 6.0 (http://www.targetscan.org), and miRanda (http://www.microrna.org) to identify potential miRNA targeting

Bioinformatics analysis reveals HNF-4A as a potential miRNA target

To address whether cellular miRNAs are involved in the regulation of HNF-4α expression, we screened the 3′-UTR of HNF-4A mRNA. Several hundred possible miRNA target sites were identified by the TargetScan database. We focused our study on the miR-34 and 449 family members, because some members of the miR-34 and miR-449 families have shown by others to be able to regulate the expression of HNF-4α [29], [31]. The families of miR-34 and miR-449 are also structurally related and share the same seed

Discussion

HNF-4α is a key member of a complex regulatory network that defines and maintains the hepatocyte phenotype. The central role of HNF-4α in liver function is highlighted by its regulation of a large number of liver specific genes [6], [7]. HNF-4α is also known to be directly and indirectly related with a number of human diseases [9], and plays a role in the hepatic response to injury [10]. The expression and activity of HNF-4α can be regulated at multiple levels. The mechanisms controlling the

Acknowledgments

This work was supported by NIH grant (R01DK064945).

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