Elsevier

Transplant Immunology

Volume 20, Issue 4, March 2009, Pages 229-231
Transplant Immunology

Brief communication
Tolerance versus immune response — MicroRNAs as important elements in the regulation of the HLA-G gene expression

https://doi.org/10.1016/j.trim.2008.11.001Get rights and content

Abstract

HLA-G is a class Ib HLA which has gained much attention due to its multiple functions on the immune system. HLA-G exerts several immunomodulatory effects, being beneficially implicated in embryo implantation and fetal survival but, conversely, being potentially detrimental in tumors and viral infections. Such a two-edged sword behavior suggest that HLA-G expression is under tight regulation. However, to date, little is known about the regulation of this gene and previous works have been unable to well correlate HLA-G regulation at the mRNA level with the polymorphic variants at the genomic level. Here we present the hypothesis that an element, which was until now neglected, might play a role in HLA-G expression regulation: MicroRNAs might participate in the regulation of the HLA-G gene expression through a putative microRNA binding site at its 3′ UTR region. Inside the 20 nt region of this microRNA binding site lies a C/G polymorphism, which was shown to be responsible for differential microRNA binding affinity and translation suppression. The role of microRNA binding on the regulation of HLA-G gene expression (and therefore on tolerance versus immune response) can be easily tested through relatively simple steps: Confirming the expression of those three complementary microRNAs in human cells which express HLA-G, followed by examination of the correlation between HLA-G mRNA and protein production controlling for HLA-G genotypes and microRNA levels; finally, selective inhibition of microRNA activity with anti-sense oligos restoring HLA-G production would access microRNA influence on HLA-G expression which, if confirmed, might help in the development of strategies to the management of several conditions in which HLA-G is involved, including pregnancy complications, transplantation, and cancer.

Introduction

HLA-G is a class Ib HLA molecule which was first characterized by its expression at the maternal–fetal interface and which has gained much attention due to its multiple functions on the immune system. As an HLA molecule, it shares structural properties of its classic counterparts HLA-A, B and C. However, unlike its counterparts, it is characterized by limited tissue distribution in healthy conditions and by the expression of seven different isoforms that can be either membrane-bound (G1–G4) or secreted (G5–G7). Since it was first described, in cytotrophoblasts, this molecule has attracted much attention due to its immunotolerogenic properties. HLA-G is capable of interacting with several receptors (ILT-2, ILT-4, KIR2DL4, CD8, CD160) present in various cells of the immune system, such as NK cells, T and B lymphocytes and antigen-presenting cells (APC). It can elicitate immune suppression by several mechanisms, such as inhibition of cytotoxicity, proliferation and/or differentiation, induction of tolerogenic APC or suppressive T and NK cells, induction of apoptosis, as well as up regulation of inhibitory receptors, among other features [reviewed in [1], [2]]. All this features have made HLA-G an attractive target in different situations in which immune tolerance is involved, such as pregnancy and its complications, transplantation, cancer and viral infections, as well as in inflammatory and autoimmune diseases [see, for example, 3]. The research in HLA-G is increasing dramatically, with over 300 references listed in the last year [4].

The fact that HLA-G exerts several immunomodulatory effects, being beneficially implicated in embryo implantation and fetal survival but, conversely, being potentially detrimental in tumors and viral infections suggests that its expression is under tight regulation. The singular organization of its promoter region, with several typical elements deleted or modified [5], reflects the HLA-G unique expression pattern among HLA molecules. This region is highly polymorphic and there are strong evidences that balancing selection might be occurring [6].

The 3′ untranslated region (UTR) seems also to have an important role on the regulation of gene expression. A 14 bp insertion/deletion polymorphism at this region has attracted the attention of many scientific groups due to its role on HLA-G alternative splicing and also on RNA stability. It was previously shown that transcripts with the 14pb sequence could undergo an additional splicing step which removes 92 bases encompassing the region in which this sequence is located. This deletion is thought to influence mRNA stability as the HLA-G transcripts with the 92 bases spliced out were shown to be more stable than the “complete” mRNAs in placental cells after actinomycin treatment [7]. Although the number of transcripts that undergo 92 bases deletion seems to vary among + 14 bp alleles and cell lineages and, in most cases, they do not represent a majority of the transcripts [7], [8], it would be expected that, overall, homozygous individuals for the insertion allele presented a higher HLA-G expression, due to the presence of these more stable transcripts. However, the proclaimed mRNA stability conferred by the 14 bp insertion seems not to have a positive effect in enhancing HLA-G expression. Instead, in heterozygote trophoblasts the measure of mRNA originated from each allele revealed that the + 14 bp allele was less expressed than the − 14 bp allele [8]. Moreover, several studies have repeatedly reported the association of the 14 bp insertion and lower soluble HLA-G levels and even the lack of detectable HLA-G expression in the plasma of homozygotes for the insertion allele [9], [10], [11], [12].

It has been shown that HLA-G expression is a fundamental pre-requisite for embryo implantation and to the maintenance of pregnancy [reviewed in 13] and the + 14/+ 14 genotype has been associated to recurrent spontaneous abortions and to the failure of in vitro fertilization [14], suggesting that this genotype could be associated to lower HLA-G levels which could, in turn, predispose to these complications. In women with pre-eclampsia, a pregnancy condition that seems to be associated to lower HLA-G production, it was observed that the placentas with the lowest HLA-G transcription levels were homozygous to the + 14 bp allele and that none of the controls of the study presented this genotype [15]. It was also reported an association between the HLA-G 14 bp insertion, as well as a particular allele (G⁎0106) containing the 14 bp insertion and preeclampsia [16], [17], [18] although controversial data come from other studies [19], [20].

So, there is evidence that, although the 14pb insertion is associated to the generation of a more stable mRNA population, these transcripts seem to have, if so, a minimal effect on overall HLA-G expression. Moreover, instead of an enhancement on HLA-G expression, a decreased protein expression is associated to the insertion allele. The existing contradiction between the stability of the transcripts originated by the + 14 alleles and the observed low HLA-G levels associated to this variant in vivo constitutes a paradox, which we name “The 14 bp polymorphism paradox”. Although this polymorphism is considered to be responsible by the alternative splicing event occurring at the 3′ in the HLA-G transcript, the lower in vivo expression associated to the + 14 bp allele is difficult to explain, even considering that transcripts lacking the 92 bases do not represent a majority of the transcripts. The answer to this paradox may involve other unknown characteristics related to this polymorphism or even other nearby polymorphisms in linkage disequilibrium (LD) with the 14 bp polymorphism.

We believe that an interesting approach to determine how the HLA-G molecule expression is regulated should include the analysis of cellular entities which are becoming very popular among research groups: MicroRNAs. MicroRNAs are small RNAs (20–22 nt) which have important and diversified functions in human biology, being involved in cell differentiation and developing, apoptosis, hematopoiesis and tumorigenesis, among other phenomena. These RNAs are able to supress gene expression when binding to specific sites at mRNA 3′ regions, through translation repression and/or by inducing RNA degradation [reviewed in 21]. At present, it is estimated that up to 30% of human genes might be regulated by microRNAs [22].

Section snippets

Are microRNAs the hidden element underlying the HLA-G 14 bp paradox?

The HLA-G gene has a microRNA binding site at its 3′ UTR region, less than 200 bp away from the 14pb polymorphic site. This site is a potential target for three microRNAs — miR-148a, miR-148b e miR-152 [23]. Inside this 20 nt region lies a C/G polymorphism, at position + 3142 (rs1063320). To test the influence of this po0lymorphism in microRNA binding, Tan and cols. performed in silico and in vitro tests which showed that the G allele favor the targeting of the three microRNAs to the binding

Evolutive considerations

When observing the homologous region of the HLA-G 3′ UTR region presented in the Pan troglodytes and Macaca mulata genome projects we observed that both species present the G allele at the position homologous for the Homo sapiens + 3142 position and the + 14 bp allele. In primates, until now, only the 14 bp insertion was observed, suggesting that it might be the ancestor allele, shared between humans and higher primates [26]. So, it is possible that the haplotype composed by the G at the + 3142

Conclusions and perspectives

Concluding, we hypothesize that the 14pb polymorphism is not the main element on the regulation of gene expression at the RNA level, although its role on alternative splicing cannot be neglected. We have exposed consistent arguments about the strong possibility of the + 3142 polymorphism as an important factor concerning HLA-G expression regulation. The rs1063320 polymorphism might influence microRNA binding to the 3′ region of the HLA-G mRNA and thus influence translation rates or even RNA

Acknowledgements

We would like to thank Iscia Lopes-Cendes for her brilliant lecture on microRNAs at the Fourth Latin American School of Human and Medical Genetics, which inspired the production of this article.

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    Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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