Brief communicationTolerance versus immune response — MicroRNAs as important elements in the regulation of the HLA-G gene expression☆
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.
References (27)
- et al.
Immuno-tolerogenic functions of HLA-G: relevance in transplantation and oncology
Autoimmun Rev
(2005) - et al.
Beyond the increasing complexity of the immunomodulatory HLA-G molecule
Blood
(2008) - et al.
The 14 bp deletion–insertion polymorphism in the 3′ UT region of the HLA-G gene influences HLA-G mRNA stability
Hum Immunol
(2003) - et al.
HLA-G expression is a fundamental prerequisite to pregnancy
Hum Immunol
(2007) - et al.
Immunogenetics of pregnancy: role of a 14-bp deletion in the maternal HLA-G gene in primiparous pre-eclamptic Brazilian women
Hum Immunol
(2007) - et al.
The HLA-G 14 bp gene polymorphism and decidual HLA-G 14 bp gene expression in pre-eclamptic and normal pregnancies
J Reprod Immunol.
(2008) - et al.
Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets
Cell
(2005) - et al.
Allele-specific targeting of microRNAs to HLA-G and risk of asthma
Am J Hum Genet.
(2007) - et al.
Evolution of MHC-G in primates: a different kind of molecule for each group of species
J Reprod Immunol.
(1999) - et al.
Evolution of MHC-G in humans and primates based on three new 3′UT polymorphisms
Hum Immunol
(2000)
Association of the HLA-G 14-bp insertion/deletion polymorphism with juvenile idiopathic arthritis and rheumatoid arthritis
Tissue Antigens
HLA-G: from biology to clinical benefits
Trends Immunol
HLA-G unique promoter region: functional implications
Immunogenetics
Cited by (95)
Lower frequency of the HLA-G UTR-4 haplotype in women with unexplained recurrent miscarriage
2018, Journal of Reproductive ImmunologyCitation Excerpt :Therefore, the exact mechanism, by which these polymorphisms may affect the occurrence of recurrent miscarriage remains to be established. Nevertheless, most polymorphisms in the 3′UTR determined in this study were previously found to be related to the extent of HLA-G expression (Hviid et al., 2003; Veit and Chies, 2009; Yie et al., 2008). Both HLA-G expressed at the fetal-maternal interface and sHLA-G possess immunosuppressive functions (Carosella et al., 2015; Le, 2015).
- ☆
Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).