Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation
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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2016, Cellular SignallingCitation Excerpt :The presence of miR-34a, miR-34c-5p, and miR-449th suppressed HNF-4α protein levels with only a small decrease in its mRNA level. The repressive effect of these miRNAs on the HNF-4α protein expression led to a significant change in the control of transcription of target genes like HNF-4α, transthyretin (TTR), apolipoprotein B (ApoB), and α1-antitrypsin (α1-AT) [98] (Table 2). Hepatocytes are the main liver forming parenchymal cells in addition to endothelial cells, cholangiocytes, Kupffer cells, stellate cells, and pit cells, which are important for the correct functioning of hepatocytes and the liver [114,115].