MiR-495 and miR-218 regulate the expression of the Onecut transcription factors HNF-6 and OC-2

doi:10.1016/j.bbrc.2009.11.052

Biochemical and Biophysical Research Communications

Volume 391, Issue 1, 1 January 2010, Pages 293-298

https://doi.org/10.1016/j.bbrc.2009.11.052 Get rights and content

Abstract

MicroRNAs are small, non-coding RNAs that posttranscriptionally regulate gene expression mainly by binding to the 3′UTR of their target mRNAs. Recent data revealed that microRNAs have an important role in pancreas and liver development and physiology. Using cloning and microarray profiling approaches, we show that a unique repertoire of microRNAs is expressed at the onset of liver and pancreas organogenesis, and in pancreas and liver at key stages of cell fate determination. Among the microRNAs that are expressed at these stages, miR-495 and miR-218 were predicted to, respectively, target the Onecut (OC) transcription factors Hepatocyte Nuclear Factor-6 (HNF-6/OC-1) and OC-2, two important regulators of liver and pancreas development. MiR-495 and miR-218 are dynamically expressed in developing liver and pancreas, and by transient transfection, we show that they target HNF-6 and OC-2 3′UTRs. Moreover, when overexpressed in cultured cells, miR-495 and miR-218 decrease the endogenous levels of HNF-6 and OC-2 mRNA. These results indicate that the expression of regulators of liver and pancreas development is modulated by microRNAs. They also suggest a developmental role for miR-495 and miR-218.

Introduction

The first sign of liver and pancreas development consists of the formation of tissue buds arising from the definitive endoderm. The progenitor cells of the pancreas and liver then proliferate and progressively differentiate to give rise to the pancreatic and hepatic cell lineages. A complex network of transcription factors drives the development of the pancreas and liver during embryogenesis [1], [2]. In particular, we have demonstrated that the Onecut transcription factors HNF-6 and OC-2 play important roles in this process [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. The analysis of knockout mice showed that HNF-6 controls the initial step of pancreas development by promoting expression of Pdx1, an essential factor for pancreas progenitor development [5]. HNF-6 and OC-2 control early pancreas morphogenesis and pancreatic duct development [9], [13]. They also promote endocrine differentiation by activating the pro-endocrine gene NGN3 [4], [9]. HNF-6 and OC-2 stimulate early liver expansion [12] and are required for normal differentiation of hepatoblasts to hepatocytes and cholangiocytes [3], [14]. Gain-of-function studies revealed that overexpression of HNF-6 in pancreatic islets induces diabetes [15], and that overexpression in adult regenerating liver stimulates hepatocyte proliferation [16]. In addition, it has been shown that accurate levels of HNF-6 are required to determine time-specific expression of HNF-6 target genes during liver development [7], [11]. Taking together, these data suggest that a precise control of the expression level of Onecut factors is necessary for acquiring or maintaining cell type-specific characters.

MicroRNAs (miRs) are short, endogenously expressed non-coding RNAs that bind to target mRNAs, mainly at their 3′UTR. By doing so, they fine-tune the expression level and repress translation of their mRNA targets. Consequently, they emerged as novel posttranscriptional regulators of gene expression. A single miR can directly target several mRNAs, and the expression of an mRNA is controlled by several miRs [17], [18]. Multiple roles have been ascribed to miRs, including in embryonic development and disease [19]. By knocking-out Dicer, a key enzyme involved in miR maturation, it was shown that miRs have an important role, among others, in the development of pancreas and liver [16], [20], [21], [22]. In the absence of Dicer in the pancreas, the number of endocrine progenitors and beta cells is strongly decreased [20]. Another study [21] pointed out the role of Dicer in maintaining the adult pancreatic phenotype. In the liver, hepatocyte-specific knockout of Dicer promotes hepatocarcinogenesis, and interestingly, in Dicer^−/− adult liver, the expression of HNF-6 is significantly increased [22]. Our unpublished results also indicate that the absence of Dicer in pancreas and liver is associated with increased levels of HNF-6 and OC-2, further suggesting that these two factors are regulated by miRs in these organs.

The aim of the present study was to identify miRs regulating Onecut factor expression. We found that two miRs, namely miR-495 and miR-218, which are expressed throughout embryonic development of the liver and pancreas, regulate Onecut factor expression by binding to target sites in the 3′UTR of HNF-6 and OC-2, respectively.

Section snippets

Materials and methods

An extended Materials and methods section is included in the online supplementary data.

MiR profiling. miR profiling in pancreas at embryonic day (e) 14.5 and in liver at e15.5 was performed using the miRCURY LNA™ Array (Exiqon). Tissues were dissected in cold PBS, RNA was extracted using Tripure reagent (Roche) and precipitated with 3.5 volumes of 100% ethanol for 30 min at −20 °C. RNA quality was assessed using an Agilent BioAnalyser 1200 system. Pancreas and liver RNAs were hybridized on

Identification of microRNAs present in developing pancreas and liver

To identify miRs that target HNF-6 and OC-2 during liver and pancreas development, we first determined the miR expression profile in embryonic pancreas and liver by a microarray approach. We found that 307 miRs were expressed in e14.5 pancreas, and 112 miRs in e15.5 liver (Supplementary Table 1). The lower number of miRs found in liver can partially be explained by a lower number of miR probes present in the version of the microarrays used, compared to the experiment with the pancreatic miRs.

Discussion

The Onecut factors HNF-6 and OC-2 are important regulators of pancreas and liver development. The absence of these factors leads to severe defects in pancreatic and liver cell differentiation [3], [4], [5], [6], [7], [8], [9], [10], [11], [13], [14]. Inversely, increased or ectopic expression is associated with perturbed cell homeostasis [16] or with diabetes [15]. This indicates that the concentration of HNF-6 and OC-2 has to be finely regulated, possibly by miRs. This hypothesis is supported

Acknowledgments

We thank D. Melton for mice, A. Tonon and Pierre Coulie for help with FACS sorting, Valérie Smekens for help, and members of the laboratory for discussions. The research was supported by grants from the 6th EU Framework Program (BetaCellTherapy Integrated Project) and from the Interuniversity Attraction Poles Program (Belgian Science Policy) to F.L., and from the Télévie and the Fund for Scientific Research to P.J. I.L. holds a Ph.D. fellowship from FRS-FNRS Télévie, P.P.P. is a Postdoctoral

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MiR-495 and miR-218 regulate the expression of the Onecut transcription factors HNF-6 and OC-2

Abstract

Introduction

Section snippets

Materials and methods

Identification of microRNAs present in developing pancreas and liver

Discussion

Acknowledgments

Gastroenterology

Dev. Biol.

J. Biol. Chem.

Dev. Biol.

Gene Expr. Patterns

Dev. Biol.

Gastroenterology

Gastroenterology

Cell

Cell

FEBS Lett.

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

Genes controlling pancreas ontogeny

Int. J. Dev. Biol.

The Onecut transcription factor HNF6 is required for normal development of the biliary tract

Development

Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3

Mol. Cell. Biol.

Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3alpha/Foxa1 gene by TGF-beta in mouse liver

Hepatology