Exosome-delivered microRNAs of “chromosome 19 microRNA cluster” as immunomodulators in pregnancy and tumorigenesis

Background Structural rearrangements of chromosomal band 19q13 are a non-random cytogenetic abnormality in thyroid adenomas and adenomatous goiters and lead to an expression of miRNAs of the chromosome 19 microRNA cluster C19MC. Normally, expression of these miRNAs is silenced except for embryonic stem cells and the placenta where they represent the majority of miRNAs not only in the trophoblast but also in exosomes derived from it. Presentation of the hypothesis We have advanced the hypothesis that as part of the feto-maternal communication miRNAs of C19MC serve immunomodulatory functions in the placenta and confer a growth advantage to thyroid nodules by protecting them against autoimmune attacks. More precisely, the exosomes containing these miRNAs may specifically target immune cells in their local environment as well as systemically by transferring their cargo to recipient cells. Within these target cells the transferred miRNAs can interact with mRNAs of the recipient cells thereby suppressing their immune-specific functions. Testing the hypothesis Experiments used to demonstrate the immunomodulatory capacity of placenta-derived exosomes can be modified by transfecting the target cells with those miRNAs of C19MC represented in placental exosomes. Implications of the hypothesis Mimics of C19MC-derived miRNAs might develop to useful drug candidates for the treatment of autoimmune disease as e.g. rheumatoid arthritis and Sjögren’s syndrome and for the prevention of transplant rejection. In case of tumor entities with elevated expression of C19MC miRNAs these miRNAs may be interesting targets for treatment with appropriate antagonists.


Background
First being described in 1989 [1], structural rearrangements of chromosomal band 19q13 are a frequent nonrandom cytogenetic abnormality in thyroid adenomas and adenomatous goiters. By applying molecular-cytogenetic methods on established cell lines it was possible to narrow down the breakpoints to a region of about 150 kbp [2] which was later shown to harbor C19MC (chromosome 19 microRNA cluster), the largest human microRNA cluster at all [3] (Figure 1). As a rule, by the chromosomal rearrangements the microRNAs of this cluster and the neighboring miR-371-3 cluster become strongly upregulated [4]. Of these both clusters C19MC is remarkable not only because of its sheer size encoding more than 50 mature microRNAs but also because of its "young" age. The whole cluster is primate-specific [3] and thus must have evolved within a relatively short time in terms of evolution. Normally, the miRNAs of C19MC are expressed almost exclusively in embryonic stem cells [5][6][7][8][9] and, later during embryonic and fetal development, only in the placenta [3,10]. Luo et al. [11] demonstrated that the trophoblast secretes exosomes, i.e. small membrane microvesicles, which contain placenta-specific miRNAs. Moreover, it was demonstrated quite recently that the vast majority of miR-NAs packed into placenta-derived exosomes consist of miRNAs of the C19MC cluster [12]. However, in both cases, i.e. in the placenta or after chromosomal translocations in benign thyroid nodules, the exact mechanisms by which C19MC miRNAs contribute to normal or aberrant functions, respectively, remain obscure.
Herein, we shall advance the hypothesis that as part of the feto-maternal communication miRNAs of C19MC serve immunomodulatory functions in the placenta and, once re-expressed due to chromosomal translocations, confer a growth advantage to thyroid nodules by protecting them against autoimmune attacks.

Presentation of the hypothesis
Though cytogenetic aberrations of 19q13.4 as detected in benign thyroid lesions represent one of the most common specific chromosomal alterations in epithelial tumors at all the molecular mechanisms resulting from these frequent genomic alterations still remain obscure. As to tumorigenesis, the stimulation of invasive growth has been attributed to some miRNAs of C19MC [for review see 13] but the activation of the cluster in thyroid adenomas and nodular goiters with 19q13.4 alterations apparently does not coincide with invasive growth. Thus, it seems unlikely that in general activation of C19MC leads to invasive growth or other features characterizing malignant cells. On the other hand, presumed "physiological functions" of its miRNAs might give us a clue to understand their role in tumorigenesis. Of note, until birth expression of C19MC persists only in the placenta or, more precisely, its trophoblast [10,11], and is expressed exclusively from the paternal allele whereas the maternal allele is silenced by epigenetic modification [14].
In general, miRNAs do not necessarily exert their main functions in the cells they are expressed in since considerable amounts of miRNAs can become packed into microvesicles called exosomes. Exosomes are bioactive vesicles derived from endosomal membranes and involved in intercellular communication by their specific cargos of proteins, mRNAs, and miRNAs [for review see 15]. In previous reports placenta-derived exosomes have been demonstrated to interact with immune cells, e.g. resulting in suppression of T-cell signaling components [16,17]. Donker et al. [12] were able to demonstrate recently that the cellular miRNA composition of human primary trophoblast cells strongly resembled that of the exosomes secreted from these cells. In both cases those of C19MC represented the majority of mature miRNAs. Of note, six microRNAs of C19MC ranged among the top-ten exosomal miRNAs. Based on their findings the authors have assumed that these miRNAs "may play an important role in placental-maternal communication, possibly directing maternal adaptation to pregnancy." Herein, we would like to outline the hypothesis that as one major function miRNAs of C19MC prevent the embryo from being attacked by the maternal immune system. Immunologically, the embryo is considered being a semi-allograft and in case of egg donation even a full allograft [18]. Nevertheless, the embryo efficiently avoids rejection by its mother's immune system by mechanisms that are not fully understood yet [for review see 19]. Exosomes are known to share membrane characteristics with the cells they are derived from [20]. Thus, it seems tempting to assume that they act like decoy-flairs for a jet. The exosomes can specifically target immune cells in their local environment, i.e. the decidua, as well as systemically thereby transferring their cargo when melting with the membrane of recipient cells. Within these target cells the transferred miRNAs can interact with mRNAs of the recipient cells thereby modulating post-transcriptional regulation. Non-specific systemic side effects of this mechanism may be the mild immunosuppression noted during pregnancy e.g. leading to the improvement of rheumatoid arthritis [for review see 21].
Tracing back to nodular goiters and thyroid adenomas re-expression of C19MC may protect cells against autoimmune attacks. Of note, a considerable percentage of these lesions develop after a pre-existing autoimmune disease of the thyroid.
Finally, the question arises if malignant tumors can adopt this mechanism to protect themselves. 19q13 is one of the most frequent chromosomal breakpoints identified in human tumors and even if, in particular in case of complex karyotypic aberrations, the small size of chromosome 19 may have resulted in false positive identification of this breakpoint there remain a number of tumor entities where its involvement has been identified unambiguously as e.g. hamartoma of the liver [22,23]. Also, a number of recent papers point to the role of amplification (e. g. in CNS-PNET [24] and in embryonal brain tumors with ependymoblastic multilayered rosettes [25]) or undermethylation of the C19MC locus (e. g. in hepatocellular carcinomas [26,27]) in several tumor entities.

Testing the hypothesis
There are straightforward appropriate experimental approaches to test our hypothesis. Nevertheless, these approaches are time consuming because not all miRNAs of the cluster may have the capacity to modulate immune cells and because different types of immune cells have to be tested. As a first step the same experiments used to demonstrate the immunomodulatory capacity of placentaderived exosomes [e.g. 17] can be modified by transfecting the target cells with those miRNAs of C19MC highly represented in placental exosomes. Furthermore, it did not escape our attention that mesenchymal cells from the amniotic membrane have strong immunomodulatory properties, e.g. by actively suppressing T-cell proliferation induced by alloantigens [28]. So far, it is believed that at term only the trophoblast expresses C19MC miRNAs but if our hypothesis holds true one would expect that these amniotic-membrane derived cells do so as well.

Implications of the hypothesis
If the hypothesis holds true it will be not only relevant in terms of basic science but also for several clinical approaches. Mimics of C19MC-derived miRNAs, either encapsulated or not, might develop to useful drug candidates for the treatment of autoimmune disease as e.g. rheumatoid arthritis and Sjögren's syndrome. Likewise, these miRNAs may prolong the maintenance of functional allografts. On the other hand, in case of tumor entities with forced expression of C19MC miRNAs their antagonists may represent interesting alternatives for targeted treatments.