Chromosome 19 miRNA Cluster miRNA Research Articles

P-800 Best matrix match: Laminin-511 improves human trophoblast stem cell identity in vitro

Abstract Study question Can alternative basement membranes support human trophoblast stem cell (hTSC) culture and recapitulate the in vivo environment more effectively than Collagen-IV (Col-IV)? Summary answer Laminin-511 (Lam-511) is the optimal alternative xeno-free matrix that enhances the stem cell state and more closely recapitulates the in vivo hTSC niche. What is known already Our knowledge on early placentation is limited due to a historical lack of robust in vitro models. Recently, hTSCs isolated from human blastocysts and first trimester placentas were successfully cultured on Col-IV, recapitulating cytotrophoblast cell self-renewal and differentiation. Even though Col-IV has been detected in the cytotrophoblast basement membrane, previously published RNA-seq analysis does not detect transcripts encoding Col-IV in the human trophectoderm. Attachment of adhesion molecules to the ECM can have an effect on cell proliferation, differentiation, polarity and gene expression. Using the correct matrix ensures a stable cell growth, as it can influence cell behaviour and identity. Study design, size, duration This was a prospective study carried out over a 6-month period. Patient-derived hTSC lines, BTS5 and CT27, obtained from RIKEN BRC Cell Bank were cultured according to the previously published protocol (1). hTSCs were maintained on culture plates coated with Col-IV, Lam-511, laminin-521 and fibronectin. Experiments were performed with biological replicates of both cell lines (n = 4), grown in every condition. Cells grown on Col-IV were used as a positive control. Participants/materials, setting, methods hTSCs were maintained on the candidate matrices for minimum of 5 passages and then were used for subsequent experiments. hTSC identity was determined by immunofluorescence analysis for known hTSC markers, GATA2 and KRT18. qRT-PCR analyses were also performed to detect additional markers EGFR, ITGA6, TEAD4, TP63, and expression from the chromosome 19 miRNA cluster (C19MC). The self-renewal capacity was assessed by cell counting and Ki67-staining. The differentiation potential was examined by performing previously published protocols. Main results and the role of chance RNA-seq analysis identified expression of ECM ligand and receptor transcripts encoding Lam-511, laminin-521 and fibronectin, which were used as candidate matrices to test. hTSC identity was maintained in all conditions as confirmed by morphological assessment, expression of known hTSC markers at the transcript and protein level, and detection of expression of C19MC miRNAs. The highest hTSC marker expression was found in cells grown on laminins. However, interestingly it was observed that only Lam-511 was capable of supporting hTSCs for more than 15 passages. Compared to Col-IV, hTSCs grown on Lam-511 appear to be more proliferative as there was a higher cell count over a 6-day period and a higher proportion of Ki-67-positive proliferative cells (p = 0.03). Additionally, preliminary data suggests that differentiation ability of hTSCs grown on Lam-511 into syncytiotrophoblasts and extravillous cytotrophoblasts is retained. Therefore, Lam-511 is an improvement to the conventional model as it enhances the stem-cell identity and closely recapitulates the in vivo niche. It can also be used as an alternative xeno-free matrix in hTSC culture in order to overcome current supply chain issues and reduce batch-to-bath variability associated with Col-IV. Limitations, reasons for caution During the 6-month period of this study, two contaminations occurred that led to slower progression of the experiments and smaller sample size. Also, there was low efficiency of the extravillous cytotrophoblast differentiation which was due to the fact that the previously published protocol performed has not yet been optimised. Wider implications of the findings Recapitulating the in vivo niche more closely will allow a deeper understanding of early placentation and help create more accurate 3D models to study the pathomechanisms of various pregnancy complications. Furthermore, utilising the xeno-free Lam-511 as an alternative matrix is a step closer to building a GMP-compliant hTSC culture. Trial registration number Not applicable

A placental specific miRNA miR-517a-3p exerts anti-human cytomegalovirus activity

Human cytomegalovirus congenital infection is the leading non-genetic cause of fetal malformation in developed countries. There are currently no safe antivirals for use during pregnancy. Placental trophoblast cells specifically secrete exosomes containing miRNA from the chromosome 19 miRNA cluster (C19MC) which confer viral resistance to recipient cells. We show the highly expressed C19MC miRNA miR-517a-3p inhibits HCMV replication and viral protein expression in both fibroblast and trophoblast cell cultures (71.6% and 50.4% inhibition of HCMV DNA at 7 days post infection respectively; p < 0.05). This naturally occurring molecule has potential for opening-up antiviral therapeutic strategies for pregnancy.

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.

Transgenic expression of human C19MC miRNAs impacts placental morphogenesis

IntroductionThe chromosome 19 miRNA cluster (C19MC) encodes a large family of microRNAs (miRNAs) that are abundantly expressed in the placenta of higher primates and also in certain cancers. In the placenta, miRNAs from this cluster account for nearly 40% of all miRNAs present in trophoblasts. However, the function of these miRNAs in the placenta remains poorly understood. Recent observations reveal a role for these miRNAs in cell migration, and suggest that they are involved in the development and function of the human placenta. Here, we examine the placenta in transgenic mice expressing the human C19MC miRNAs. MethodsWe produced transgenic mice using pronuclear microinjection of a bacterial artificial chromosome plasmid carrying the entire human C19MC locus and derived a homozygous line using crossbreeding. We performed morphological characterization and profiled gene expression changes in the placentas of the transgenic mice. ResultsC19MC transgenic mice delivered on time with no gross malformations. The placentas of transgenic mice expressed C19MC miRNAs and were larger than wild type placentas. Histologically, we found that the transgenic placenta exhibited projections of spongiotrophoblasts that penetrated deep into the labyrinth. Gene expression analysis revealed alterations in the expression of several genes involved in cell migration, with evidence of enhanced cell proliferation. DiscussionMice that were humanized for transgenically overexpressed C19MC miRNAs exhibit enlarged placentas with aberrant delineation of cell layers. The observed phenotype and the related gene expression changes suggest disrupted migration of placental cell subpopulations.

Unc-13 homolog D mediates an antiviral effect of the chromosome 19 microRNA cluster miR-517a.

The function of microRNAs (miRNAs) can be cell autonomous or communicated to other cell types and has been implicated in diverse biological processes. We previously demonstrated that miR-517a-3p (miR-517a), a highly expressed member of the chromosome 19 miRNA cluster (C19MC) that is transcribed almost exclusively in human trophoblasts, attenuates viral replication via induction of autophagy in non-trophoblastic recipient cells. However, the molecular mechanisms underlying these effects remain unknown. Here, we identified unc-13 homolog D (UNC13D) as a direct, autophagy-related gene target of miR-517a, leading to repression of UNC13D. In line with the antiviral activity of miR-517a, silencing UNC13D suppressed replication of vesicular stomatitis virus (VSV), whereas overexpression of UNC13D increased VSV levels, suggesting a role for UNC13D silencing in the antiviral activity of miR-517a. We also found that miR-517a activated NF-κB signaling in HEK-293XL cells expressing TLR8, but the effect was not specific to C19MC miRNA. Taken together, our results define mechanistic pathways that link C19MC miRNA with inhibition of viral replication.

B-101 Placental exosomes in maternal-placental-fetal communication and viral resistance

The placenta plays a critical role in regulating maternal-fetal gas exchange, supply of nutrients to the fetus and removal of waste, production of hormones, and immunological defense, including resistance to diverse pathogens. While many of these functions are controlled by hormones, growth factors and other signaling molecules, the recent discovery of placental small RNAs and their release to the plasma within extracellular vesicles provides a new dimension to our investigation into paracrine and distant signaling networks that constitute maternal-fetal crosstalk. In this context, we found that primary human trophoblasts are resistant to infection by diverse types of DNA and RNA viruses, and that this resistance can be partly conferred to non-trophoblastic cells by transferring placenta- and primate-specific miRNAs, expressed from the chromosome 19 miRNA cluster (C19MC). Furthermore, we found that all trophoblastic EVs contain C19MC miRNAs, and that trophoblastic exosomes execute an antiviral effect when applied to non-placental cells. To pursue the patterns of miRNA transport in vivo among the maternal, placental and fetal compartments, we created a humanized mouse model that stably expresses the human C19MC miRNA cluster. We detected the trafficking of placental miRNAs among the maternal and fetal compartments. Our current work centers on the mechanisms of uptake of trophoblastic exosomes by non-trophoblastic cells, and the proteins that are essential for that process. Together, our findings establish previously unrecognized mechanisms of exosome-based maternal-fetal crosstalk. These pathways have functional implications for defense against viral pathogens and possibly have other homeostatic functions during pregnancy.

Chromosome 19 miRNA cluster and CEBPB expression specifically mark and potentially drive triple negative breast cancers.

Triple negative breast cancers (TNBCs) are known to express low PGR, ESR1, and ERBB2, and high KRT5, KRT14, and KRT17. However, the reasons behind the increased expressions of KRT5, KRT14, KRT17 and decreased expressions of PGR, ESR1, and ERBB2 in TNBCs are not fully understood. Here we show that, expression of chromosome 19 miRNA cluster (C19MC) specifically marks human TNBCs. Low REST and high CEBPB correlate with expression of C19MC, KRT5, KRT14, and KRT17 and enhancers of these genes/cluster are regulated by CEBPB and REST binding sites. The C19MC miRNAs in turn can potentially target REST to offer a positive feedback loop, and might target PGR, ESR1, ERBB2, GATA3, SCUBE2, TFF3 mRNAs to contribute towards TNBC phenotype. Thus our study demonstrates that C19MC miRNA expression marks TNBCs and that C19MC miRNAs and CEBPB might together determine the TNBC marker expression pattern.

MiR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming

BackgroundIntroduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to ‘reprogram’ somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.MethodsA miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.ResultsCo-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.ConclusionsVia targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

The expression level of C19MC miRNAs in early pregnancy and in response to viral infection

IntroductionWe have previously shown that miRNAs produced from the Chromosome 19 MiRNA Cluster (C19MC), which are expressed almost exclusively in primate trophoblasts and are released into the maternal circulation, reduce viral replication in non-placental cells and can modulate migratory behavior of extravillous trophoblast. We sought to define the expression pattern of C19MC miRNA in early pregnancy and in response to viral infection in vitro and in vivo. MethodsWe prospectively followed women undergoing in vitro fertilization (IVF) and determined their blood level of C19MC miRNA using RT-qPCR. To examine the effect of viral exposure on C19MC miRNAs expression, we used three systems: (1) a transgenic mouse overexpressing the C19MC cluster and exposed to Togaviridae during pregnancy, (2) cultured primary human trophoblasts exposed to Vesicular Stomatitis Virus in vitro, and (3) amniotic fluid from women exposed to cytomegalovirus during pregnancy. ResultsIn 27 IVF pregnancies, C19MC miRNAs were detected as early as 2 weeks after implantation, and their levels increased thereafter. There was no change in C19MC miRNA expression levels in the mouse placenta in response to viral exposure. Similarly, Vesicular Stomatitis Virus infection of primary human trophoblast did not selectively increase C19MC miRNA expression. C19MC miRNA expression in the amniotic fluid was not affected by vertical transmission of cytomegalovirus. DiscussionThe expression of C19MC miRNAs in maternal circulation very early in pregnancy suggests a role in the establishment of the maternal-fetal interface. The levels of C19MC miRNA are not influenced by diverse types of viral infection.

Expression and trafficking of placental microRNAs at the feto-maternal interface.

During pregnancy, placental trophoblasts at the feto-maternal interface produce a broad repertoire of microRNA (miRNA) species. These species include miRNA from the primate-specific chromosome 19 miRNA cluster (C19MC), which is expressed nearly exclusively in the placenta. Trafficking of these miRNAs among the maternal, placental, and fetal compartments is unknown. To determine miRNA expression and trafficking patterns during pregnancy, we sequenced miRNAs in triads of human placenta and of maternal and fetal blood and found large subject-to-subject variability, with C19MC exhibiting compartment-specific expression. We therefore created humanized mice that transgenically express the entire 160-kb human C19MC locus or lentivirally express C19MC miRNA members selectively in the placenta. C19MC transgenic mice expressed a low level of C19MC miRNAs in diverse organs. When pregnant, female C19MC mice exhibited a strikingly elevated (>40-fold) expression of C19MC miRNA in the placenta, compared with other organs, that resembled C19MC miRNAs patterns in humans. Our mouse models showed that placental miRNA traffic primarily to the maternal circulation and that maternal miRNA can traffic to the placenta and even into the fetal compartment. These findings define an extraordinary means of nonhormonal, miRNA-based communication between the placenta and feto-maternal compartments.-Chang, G., Mouillet, J.-F., Mishima, T., Chu, T., Sadovsky, E., Coyne, C. B., Parks, W. T., Surti, U., Sadovsky, Y. Expression and trafficking of placental microRNAs at the feto-maternal interface.

Selective activation of miRNAs of the primate-specific chromosome 19 miRNA cluster (C19MC) in cancer and stem cells and possible contribution to regulation of apoptosis

BackgroundThe human chromosome 19 miRNA cluster (C19MC) of 43 genes is a primate-specific miRNA cluster that may have biological significance in the genetic complexity of the primate. Despite previous reports on individual C19MC miRNA expression in cancer and stem cells, systematic studies on C19MC miRNA expression and biological functions are lacking.ResultsCluster-wide C19MC miRNA expression profiling by microarray analysis showed wholesome C19MC activation in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, in multipotent adipose-derived mesenchymal stem cells (MSCs) and a unipotent human white pre-adipocyte cell line, only selected C19MC miRNAs were expressed. MiRNA copy number analysis also showed selective C19MC expression in cancer cells with expression patterns highly similar to those in MSCs, suggesting similar miRNA regulatory mechanisms in these cells. Selective miRNA expression also suggests complex transcriptional mechanism(s) regulating C19MC expression under specific cellular and pathological conditions. Bioinformatics analysis showed that sixteen of the C19MC miRNAs share the same “AAGUGC” seed sequence with members of the miR-302/-372 family, which are known cellular reprogramming factors. In particular, C19MC-AAGUGC-miRNAs with the nucleotides 2-7 canonical seed position as in miR-302/-372 miRNAs, may play similar roles as miR-302/-372 in induced pluripotency. A biased 3p-arm selection of the C19MC-AAGUGC-miRNAs was observed indicating that targets of the 3p species of these miRNAs may be biologically significant in regulating stemness. Furthermore, bioinformatics analysis of the putative targets of the C19MC-AAGUGC-miRNAs predicted significant involvement of signaling pathways in reprogramming, many of which contribute to promoting apoptosis by indirect activation of the pro-apoptotic proteins BAK/BAX via suppression of genes of the cell survival pathways, or by enhancing caspase-8 activation through targeting inhibitors of TRAIL-inducing apoptosis.ConclusionsThis work demonstrated selective C19MC expression in MSCs and cancer cells, and, through miRNA profiling and bioinformatics analysis, predicted C19MC modulation of apoptosis in induced pluripotency and tumorigenesis.

Coexpression of TrkB and PD-L1 on human placenta

Objective: We performed comprehensive miRNA and gene expression profiling of maternal peripheral blood natural killer (pNK) cells during pregnancy. Methods: Samples of pNK cells at different time points of pregnancy (first, second, and third trimester of gestation, and postdelivery) were obtained from healthy pregnant females with their informed consent. We performed real-time PCR-based array analysis to examine the expression levels of 756 miRNAs in maternal pNK cells using TaqMan Array MicroRNA Cards. Moreover, we performed DNA microarray analysis of gene expression levels in firstand third-trimester pNK cells using Agilent Microarray Kits. Results: PCR-based array analysis showed transfer of placentaderivedmiRNAs [chromosome19miRNAcluster (C19MC)miRNAs] into maternal pNK cells occurred during pregnancy. Rapid clearance of C19MC miRNAs also occurred in pNK cells after delivery. DNA microarray analysis identified NK cell function-related genes (e.g., KIR genes) that were differentially expressed between firstand third-trimester pNK cells. Conclusions: Maternal pNK cells may adjust to different gestational conditions via the uptake of exogenousmiRNAs (e.g., C19MC miRNAs) during pregnancy.

Analysis of miRNA-mRNA expression profiles in maternal peripheral blood natural killer cells during pregnancy

Objective: We performed comprehensive miRNA and gene expression profiling of maternal peripheral blood natural killer (pNK) cells during pregnancy. Methods: Samples of pNK cells at different time points of pregnancy (first, second, and third trimester of gestation, and postdelivery) were obtained from healthy pregnant females with their informed consent. We performed real-time PCR-based array analysis to examine the expression levels of 756 miRNAs in maternal pNK cells using TaqMan Array MicroRNA Cards. Moreover, we performed DNA microarray analysis of gene expression levels in firstand third-trimester pNK cells using Agilent Microarray Kits. Results: PCR-based array analysis showed transfer of placentaderivedmiRNAs [chromosome19miRNAcluster (C19MC)miRNAs] into maternal pNK cells occurred during pregnancy. Rapid clearance of C19MC miRNAs also occurred in pNK cells after delivery. DNA microarray analysis identified NK cell function-related genes (e.g., KIR genes) that were differentially expressed between firstand third-trimester pNK cells. Conclusions: Maternal pNK cells may adjust to different gestational conditions via the uptake of exogenousmiRNAs (e.g., C19MC miRNAs) during pregnancy.

C19MC microRNAs regulate the migration of human trophoblasts.

Early in pregnancy, trophoblast invasion into the decidua and inner myometrium is essential for establishment of proper implantation, maternal-fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts (VTs), extravillous trophoblasts (EVTs) are unique in their capacity to invade the maternal decidua and myometrium. The largest human microRNA (miRNA) gene cluster, the chromosome 19 miRNA cluster (C19MC), is expressed almost exclusively in the placenta and, rarely, in certain tumors and undifferentiated cells. In the work reported here, we found that the expression of C19MC miRNAs is higher in VTs than in EVTs. Using a bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line, which does not naturally express these miRNAs, we found that C19MC miRNAs selectively attenuate cell migration without affecting cell proliferation or apoptosis. A microarray analysis revealed that C19MC miRNAs regulate target transcripts related to cellular movement. Our data also implicated a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype by targeting CXCL6, NR4A2 and FOXL2 transcripts through a 3'UTR miRNA-responsive element. Together, our data suggest a role for C19MC miRNAs in modulating the migration of EVTs.

Placenta‐specific miRNA (miR‐512‐3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells

The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets. A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay. By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system. These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.

Small but Effective: Trophoblast-Derived miRNAs Transported Via Exosomes as Guardians Against Viral Infections

Evaluation of: Delorme-Axford E, Donker RB, Mouillet JF et al. Human placental trophoblasts confer viral resistance to recipient cells. Proc. Natl Acad. Sci. USA 110(29), 12048–12053 (2013). Chromosome 19 miRNA cluster (C19MC) is the largest human miRNA cluster. It is almost exclusively expressed in extraembryonic tissue, particularly of the placenta. Its miRNAs may not only act locally, but, being released to the maternal circulation in large amounts, may also affect other tissues. Recently, Delorme-Axford et al. showed that C19MC miRNAs contribute to viral resistance in the trophoblast and confer this resistance to several other cells via exosomes. Furthermore, they provide strong evidence that this antiviral activity is based on the induction of autophagy in the target cells. These findings indicate a pivotal role of C19MC miRNAs in pregnancy by counteracting embryonal and fetal infections and, simultaneously, raise several further questions, including therapeutic approaches.

MicroRNA profiling of hepatocarcinogenesis identifies C19MC cluster as a novel prognostic biomarker in hepatocellular carcinoma

Progressive hepatocarcinogenesis is a stepwise process that drives liver transformation. However, the molecular mechanisms of early liver transformation are far from clear. A role for microRNAs (miRNA) as diagnostic and prognostic factors in human tumours, including hepatocellular carcinoma (HCC), is promising. We aimed to identify novel miRNA as biomarkers for differential diagnosis and predictors of disease progression. We used a low-density array platform to profile the expression of 664 mature miRNA in a cohort of 60 hepatitis C virus-positive liver lesions representative of all stages of progressive hepatocarcinogenesis. We validated selected miRNA in two independent patient series by qPCR and we characterized the genomic status of the miRNA cluster C19MC by fluorescent in situ hybridization and copy-number variation analyses. A 18-miRNA signature distinguished cirrhosis, dysplastic nodules and HCC lesions. Four miRNAs overexpressed in HCCs belonged to chromosome 19 miRNA cluster (C19MC). Significant overexpression of C19MC in early HCC compared to dysplastic nodules could be confirmed in a second series of hepatitis B virus-related liver lesions (n=30). In a third series of 61 HCCs, C19MC cluster was overexpressed in HCCs compared to matched cirrhotic parenchyma and regardless of the type of viral infection. High C19MC miRNA levels were correlated with poor clinico-pathological features, increased risk of tumour recurrence and shorter overall survival time. HCCs overexpressing the C19MC cluster showed genetic amplification of the corresponding locus. C19MC cluster is a novel molecular alteration characteristic of liver cancer and predictor of poor prognosis. C19MC is an attractive candidate for novel HCC therapies.

The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes

The largest gene cluster of human microRNAs (miRNAs), the chromosome 19 miRNA cluster (C19MC), is exclusively expressed in the placenta and in undifferentiated cells. The precise expression pattern and function of C19MC members are unknown. We sought to profile the relative expression of C19MC miRNAs in primary human trophoblast (PHT) cells and exosomes. Using high-throughput profiling, confirmed by PCR, we found that C19MC miRNAs are among the most abundant miRNAs in term human trophoblasts. Hypoxic stress selectively reduced miR-520c-3p expression at certain time-points with no effect on other C19MC miRNAs. Similarly, differentiation in vitro had a negligible effect on C19MC miRNAs. We found that C19MC miRNAs are the predominant miRNA species expressed in exosomes released from PHT, resembling the profile of trophoblastic cellular miRNA. Predictably, we detected the similar levels of circulating C19MC miRNAs in the serum of healthy pregnant women at term and in women with pregnancies complicated by fetal growth restriction. Our data define the relative expression levels of C19MC miRNAs in trophoblasts and exosomes, and suggest that C19MC miRNAs function in placental-maternal signaling.

RNA polymerase III transcribes human microRNAs

Prior work demonstrates that mammalian microRNA (miRNA or miR) expression requires RNA polymerase II (Pol II). However, the transcriptional requirements of many miRNAs remain untested. Our genomic analysis of miRNAs in the human chromosome 19 miRNA cluster (C19MC) revealed that they are interspersed among Alu repeats. Because Alu transcription occurs through RNA Pol III recruitment, and we found that Alu elements upstream of C19MC miRNAs retain sequences important for Pol III activity, we tested the promoter requirements of C19MC miRNAs. Chromatin immunoprecipitation and cell-free transcription assays showed that Pol III, but not Pol II, is associated with miRNA genomic sequence and sufficient for transcription. Moreover, the mature miRNA sequences of approximately 50 additional human miRNAs lie within Alu and other known repetitive elements. These findings extend the current view of miRNA origins and the transcriptional machinery driving their expression.