miRNA Population Research Articles

Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

Abstract 5033: Targeted delivery of miR-122 into hepatocytes regresses liver metastasis

Abstract Background: Metastatic tumors in the liver are 20 times more common than primary tumors and considered as a leading cause of mortality in several types of cancer. Therefore, making the liver (soil) inhospitable for tumor cells (seeds) to grow will have tremendous effects on outcome of multiple types of cancer. RNAi delivery -including miRNA delivery-shows promise for the development of novel molecular therapeutics that interfere with genes causing metastasis. Mir-122, which accounts for 70% of total miRNA population within the liver, is also known as a tumor suppressor miRNA within liver. We hypothesized that miR-122 delivery into hepatocytes can prevent development of metastatic colorectal cancer within mice liver. For testing this hypothesis, we used hepatic injection of ex-vivo engineered liver metastasis from human colorectal cancer cells to induce hepatic metastasis in nude mice and employed lipid calcium phosphate (LCP) nanoparticles for delivery of miR-122. Methods: LCP-miR122 and LCP-miRNC (negative control miRNA) were prepared mixing an emulsion of calcium which contained miRNA with a phosphate emulsion, followed by addition of DOPA to make the inner core of LCP. The outer leaflet of LCP contained DOTAP, Cholesterol and DSPE-PEG as. Galactose was conjugated at LCP surface for its targeted delivery to hepatocytes. For induction of hepatic metastasis, HT29-Luc cells were grown on liver biomatrix (BMX) for 7-10 days to acquire metastatic features and then harvested and injected into portal veins of nu/nu mice. Hepatic metastases formation was monitored weekly in vivo using bioluminescent imaging. LCP-miR122 or LCP-miRNC was systematically injected three times a week for 3 weeks starting 24 hours after tumor inoculations. Results: LCP particles were found to be 25-50 nm via dynamic light scattering analysis. When injected in vivo, LCP containing miR-122 was majorly distributed into liver (80%) and was detected in hepatocytes through confocal microscopy and target gene expression analysis. LCP-miR122 did not show any sign of hepatotoxicity or renal toxicity or any other pathological damage to other major organs. The injected conditioned HT29 cells formed metastatic foci within liver 1-3 weeks after portal vein injection as detected by bioluminescent imaging. Interestingly, we found that 67% of (6 out of 9) mice treated with LCP-miR122 did not show any sign of liver metastasis after 3 weeks compared with only 20% (2 out of 10) of mice treated with LCP containing negative control miRNA [OR=0.12, 95% CI:0.015-0.99; p=0.05]. Monitoring liver metastasis expansion on weekly basis, mice treated with LCP-miR122 showed a slight decrease in number of liver metastatic foci compared with control mice which showed a significant increase in number of liver metastatic foci during treatment [0.7 vs 3.8-fold; p=0.05]. Conclusion: These results suggest that targeted delivery of miR-122 into hepatocytes of patients prone to liver metastasis or harboring liver metastasis can prevent or regress hepatic metastasis. Citation Format: Hossein Sendi, Nikhila Sultanpuram, Jie Wang, Amanda Graboski, Liantao Li, Mengying Hu, Leaf Huang, Andrew Z. Wang. Targeted delivery of miR-122 into hepatocytes regresses liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5033.

Effects of endurance racing on horse plasma extracellular particle miRNA.

Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. To evaluate the effects of 160km endurance racing on horse plasma extracellular particles and their miRNA population. Cohort study. We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2hours after the race, which returned to baseline 15hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.

Optimization of small RNA library preparation protocol from human urinary exosomes

BackgroundSequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomarkers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Here, we have assessed several steps in developing an optimized small RNA (sRNA) library preparation protocol for next-generation sequencing (NGS) miRNA analysis from urinary exosomes.MethodsA total of 24 urinary exosome samples from donors were included in this study. RNA was extracted by column-based methods. The quality of extracted RNA was assessed by spectrophotometric quantification and Bioanalyzer software analysis. All libraries were prepared using the CleanTag small RNA library preparation protocol and the effect of our additional modifications on adapter-dimer presence, sequencing data and tagged small RNA library population was also analyzed.ResultsOur results show that good quality sequencing libraries can be prepared following our optimized small RNA library preparation protocol from urinary exosomes. When the size selection by gel purification step was included within the workflow, adapter-dimer was totally removed from cDNA libraries. Furthermore, the inclusion of this modification step within small RNA library protocol augmented the small RNA mapped reads, with an especially significant 37% increase in miRNA reads, and the gel purification step made no difference to the tagged miRNA population.ConclusionsThis study provides researchers with an optimized small RNA library preparation workflow for next generation sequencing based exosome-associated miRNA analysis that yields a high amount of miRNA mapped reads without skewing the tagged miRNA population significantly.

Identification of Grafting-Responsive MicroRNAs Associated with Growth Regulation in Pecan [Carya illinoinensis (Wangenh.) K. Koch

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an economically important nut tree and grafting is often used for clonal propagation of cultivars. However, there is a lack of research on the effects of rootstocks on scions, which are meaningful targets for directed breeding of pecan grafts. MicroRNAs (miRNAs) play an important role in many biological processes, but the mechanism underlying the involvement of miRNAs in grafting-conferred physiological changes is unclear. To identify the grafting-responsive miRNAs that may be involved in the regulation of growth in grafted pecan, six small RNA libraries were constructed from the phloem of two groups of grafts with significantly different growth performance on short and tall rootstocks. A total of 441 conserved miRNAs belonging to 42 miRNA families and 603 novel miRNAs were identified. Among the identified miRNAs, 24 (seven conserved and 17 novel) were significantly differentially expressed by the different grafts, implying that they might be responsive to grafting and potentially involved in the regulation of graft growth. Ninety-five target genes were predicted for the differentially expressed miRNAs; gene annotation was available for 33 of these. Analysis of their targets suggested that the miRNAs may regulate auxin transport, cell activity, and inorganic phosphate (Pi) acquisition, and thereby, mediate pecan graft growth. Use of the recently-published pecan genome enabled identification of a substantial population of miRNAs, which are now available for further research. We also identified the grafting-responsive miRNAs and their potential roles in pecan graft growth, providing a basis for research on long-distance regulation in grafted pecan.

Identification of novel microRNAs and their targets in Chlorophytum borivilianum by small RNA and degradome sequencing.

AbstarctPlant specific miRNAs (Novel miRNAs) are well known to perform distinctive functions in biological processes. Identification of new miRNAs is necessary to understand their gene regulation. Degradome provides an opportunity to explore the miRNA functions by comparing the miRNA population and their degraded products. In the present study, Small RNA sequencing data was used to identify novel miRNAs. Further, degradome sequencing was carried out to identify miRNAs targets in the plant, Chlorophytum borivilianum. The present study supplemented 40 more novel miRNAs correlating degradome data with smallRNAome. Novel miRNAs, complementary to mRNA partial sequences obtained from degradome sequencing were actually targeting the later. A big pool of miRNA was established by using Oryza sativa, Arabidopsis thaliana, Populus trichocarpa, Ricinus communis, and Vitis vinifera genomic data. Targets were identified for novel miRNAs and total 109 targets were predicted. BLAST2GO analysis elaborate about localization of novel miRNAs’ targets and their corresponding KEGG (Kyoto Encyclopedia for Genes and Genomes) pathways. Identified targets were annotated and were found to be involved in significant biological processes like Nitrogen metabolism, Pyruvate metabolism, Citrate cycle (TCA cycle), photosynthesis, and Glycolysis/Gluconeogenesis. The present study provides an overall view of the miRNA regulation in multiple metabolic pathways that are involved in plant growth, pathogen resistance and secondary metabolism of C. borivilianum.

The role of miRNAs in male human reproduction: a systematic review.

The presence of miRNAs in human reproductive tissue is intriguing and suggests the possibility that these important regulatory molecules play a role in reproductive function. However, the regulatory role of miRNAs in reproductive tissue remains poorly understood with a significant amount of controversial and contradicting data. To systematically review the high-quality studies published to date investigating miRNAs associated with male human reproduction in order to describe their roles and relations with infertility and update the knowledge in the field. A comprehensive systematic review of the published literature in MEDLINE-PubMed and EMBASE databases from the earliest available online indexing year until June 2018 (complimentary search until July 2019) was performed, in accordance with the PRISMA guidelines. We have included descriptive, case-control, cross-sectional, and observational prospective and retrospective studies in which fertile/infertile men were well-defined. The primary outcome was the miRNA expression in testis, epididymis, sperm cells, seminal plasma, and extracellular vesicles (i.e., exosomes and microvesicles). We identified 25,204 articles, of which 42 were selected for qualitative analysis. Of the 42 articles included, 15 evaluated the miRNAs in testis, five in epididymis, 13 in spermatozoa, and 11 in seminal plasma and/or extracellular vesicles. Two studies tackled more than one sub-group. As far as miRNA presence and content, the results of this systematic review indicated that every tissue/cell contains a well-defined and stable population of miRNAs that could be potentially related to spermatogenesis and embryogenesis. Our systematic review of descriptive and observational studies shows a consistent relationship between aberrant miRNA expression and infertility. Therefore, it seems reasonable that measuring the expression of particular miRNAs might be useful not only as infertility biomarkers, but also for developing therapeutic strategies.

Organ-specific small non-coding RNA responses in domestic (Sudani) ducks experimentally infected with highly pathogenic avian influenza virus (H5N1)

ABSTRACT The duck represents an important reservoir of influenza viruses for transmission to other avian and mammalian hosts, including humans. The increased pathogenicity of the recently emerging clades of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype in ducks features systemic viral spread and organ-to-organ variation in viral transcription and tissue damage. We previously reported that experimental infection of Sudani ducks (Cairina moschata) with an Egyptian HPAI (H5N1) virus (clade 2.2.1.2) features high viral replication and severe tissue damage in lung, but lower viral replication and only mild histological changes in brain. Little is known about the involvement of miRNA in organ-specific responses to H5N1 viruses in ducks, and involvement of the other classes of small noncoding RNA (sncRNA) has not been investigated so far. Following RNA sequencing, we have annotated the duck sncRNome and compared global expression changes of the four major sncRNA classes (miRNAs, piRNAs, snoRNAs, snRNAs) between duck lung and brain during a 120 h time course of infection with this HPAI strain. We find major organ-specific differences in miRNA, piRNA and snoRNA populations even before infection and substantial reprogramming of all sncRNA classes throughout infection, which was less pronounced in brain. Pathway prediction analysis of miRNA targets revealed enrichment of inflammation-, infection- and apoptosis-related pathways in lung, but enrichment of metabolism-related pathways (including tryptophan metabolism) in brain. Thus, organ-specific differences in sncRNA responses may contribute to differences in viral replication and organ damage in ducks infected with isolates from this emerging HPAI clade, and likely other strains.

Rolling circle amplification and graphene-based sensor-on-a-chip for sensitive detection of serum circulating miRNAs

In this study, we developed a simple multiplex miRNA detection platform based on rolling circle amplification and the fluorescence quenching property of reduced graphene oxide. The detection platform could be applied on a microfluidics chip with a mobile system controller to eliminate contamination and to facilitate potential use in remote areas. As a proof of concept, two fluorescence-labeled ssDNA tags were used for detection of miR-29a and miR-144*, two miRNAs that are highly expressed in the blood circulation of some patients with cancer or tuberculosis. The circular ssDNA probes in this study were designed to have an advantage over padlock probes as they can be prepared in advance. Our multiplex miRNA detection platform exhibited high sensitivity and selectivity, with a limit of detection of 0.05 pmol. In addition, our platform could detect target miRNAs from the total miRNA population extracted from human serum or a cancer cell line. These results indicated that our miRNA sensor has the potential to provide simple and high throughput miRNA analysis for disease diagnosis and prognosis.

Computational prediction of microRNAs in marine bacteria of the genus Thalassospira.

MicroRNAs (miRNAs) are key players in regulation of gene expression at post-transcription level in eukaryotic cells. MiRNAs have been intensively studied in plants, animals and viruses. The investigations of bacterial miRNAs have gained less attention, except for the recent studies on miRNAs derived from Streptococcus mutans ATCC 25175 and Escherichia coli DH10B. In this study, high-throughput sequencing approach was employed to investigate the miRNA population in bacteria of the genus Thalassospira using both the miRDeep2 and CID-miRNA methods. A total of 984 putative miRNAs were identified in 9 species of the genus Thalassospira using both miRDeep and CID-miRNA analyses. Fifty seven conserved putative miRNAs were found in different species of the genus Thalassospira, and up to 6 miRNAs were found to be present at different locations in the T. alkalitolerans JCM 18968T, T. lucentensis QMT2T and T. xianhensis P-4T. None of the putative miRNAs was found to share sequence to the reported miRNAs in E. coli DH10B and S. mutans ATCC 25175. The findings provide a comprehensive list of computationally identified miRNAs in 9 bacterial species of the genus Thalassospira and addressed the existing knowledge gap on the presence of miRNAs in the Thalassospira genomes.

Hotspot Mutations in DICER1 Causing GLOW Syndrome-Associated Macrocephaly via Modulation of Specific microRNA Populations Result in the Activation of PI3K/ATK/mTOR Signaling.

We have previously described mosaic mutations in the RNase IIIb domain of DICER1that display global developmental delays, lung cysts, somatic overgrowth, macrocephaly and Wilms tumor. This constellation of phenotypes was classified as GLOW syndrome. Due to the phenotypic overlap between GLOW and syndromes caused by mutations in the PI3K/AKT/mTOR pathway, we hypothesized that alterations in miRNA regulation of this pathway cause its specific constellation of phenotypes. To test the hypothesis that DICER1 "hot spot" mutations associated with GLOW syndrome activate PI3K/AKT/mTOR signaling. We developed HEK293T cells with loss of exon 25 in DICER1, a genetic modification that is synonymous with the "hot spot" RNAseIIIb mutations that cause GLOW syndrome. We assayed the cells for activation of the PI3K/AKT/mTOR signaling pathway. We observed activation of the PI3K/AKT/mTOR pathway as demonstrated by increased pS6Kinase, p4EBP1 and pTSC2 levels. Additionally, these cells demonstrate a striking cellular phenotype, with the ability to form spheres when the serum is removed from their growth medium. The cells in these spheres are Oct4 and Sox2 positive and exhibit the property of reversion with the addition of serum. We queried miRNA expression data and identified a population of miRNAs that increase due to these mutations and target negative regulators of the PI3K/AKT/mTOR pathway. This work identifies the delicate and essential role for miRNA control of the PI3K/AKT/mTOR pathway. We conclude that the phenotypes observed in the GLOW syndrome are the result of PI3K/AKT/mTOR activation.

P73-Governed miRNA Networks: Translating Bioinformatics Approaches to Therapeutic Solutions for Cancer Metastasis.

The transcription factor p73 synthesizes a large number of isoforms and presents high structural and functional homology with p53, a well-known tumor suppressor and a famous "Holy Grail" of anticancer targeting. p73 has attracted increasing attention mainly because (a) unlike p53, p73 is rarely mutated in cancer, (b) some p73 isoforms can inhibit all hallmarks of cancer, and (c) it has the ability to mimic oncosuppressive functions of p53, even in p53-mutated cells. These attributes render p73 and its downstream pathways appealing for therapeutic targeting, especially in mutant p53-driven cancers. p73 functions are, at least partly, mediated by microRNAs (miRNAs), which constitute nodal components of p73-governed networks. p73 not only regulates transcription of crucial miRNA genes, but is also predicted to affect miRNA populations in a transcription-independent manner by developing protein-protein interactions with components of the miRNA processing machinery. This combined effect of p73, both in miRNA transcription and maturation, appears to be isoform-dependent and can result in a systemic switch of cell miRNomes toward either an anti-oncogenic or oncogenic outcome. In this review, we combine literature search with bioinformatics approaches to reconstruct the p73-governed miRNA network and discuss how these crosstalks may be exploited to develop next-generation therapeutics.

Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF mutated melanoma

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.

The maternal-to-zygotic transition in bovine in vitro-fertilized embryos is associated with marked changes in small non-coding RNAs†

In mammals, small non-coding RNAs (sncRNAs) have been reported to be important during early embryo development. However, a comprehensive assessment of the inventory of sncRNAs during the maternal-to-zygotic transition (MZT) has not been performed in an animal model that better represents the sncRNA biogenesis pathway in human oocytes and embryos. The objective of this study was to examine dynamic changes in expression of sncRNAs during the MZT in bovine embryos produced by in vitro fertilization (IVF), which occurs at the 8-cell stage. An unbiased, discovery-based approach was employed using small RNAseq to profile sncRNAs in bovine oocytes, 8-cell stage embryos and blastocyst stage embryos followed by network and ontology analyses to explore the functional relevance of differentially expressed micro-RNAS (miRNAs). The relative abundance of miRNAs was markedly higher in 8-cell stage embryos compared to oocytes or blastocyst stage embryos. This shift in miRNA population was largely associated with upregulation of miRNAs predicted to target genes involved in the biological processes of cell development, cell division, Wnt signaling, and pluripotency, among others. Distinct populations of piwi-interacting-like RNAs (pilRNAs) were identified in bovine oocytes and blastocyst stage embryos, though pilRNAs were nearly absent in 8-cell stage embryos. Also, small nucleolar RNAs were highly expressed in 8-cell stage embryos. Overall, these data reveal a strong dynamic shift in the relative abundance of sncRNAs associated with the MZT in bovine oocytes and embryos, suggesting that these molecules may play important roles in the shift from maternal to zygotic control of gene expression.

Feasibility of microRNA profiling in human inner ear perilymph.

Hearing loss is common and caused by a wide range of molecular and cellular pathologies. Current diagnosis of hearing loss depends on a combination of physiologic testing, patient history, and in some cases genetic testing. Currently, no biopsy or equivalent procedure exists to diagnose inner ear disorders. MicroRNAs (miRNA) are short ribonucleic acids that regulate a variety of cellular processes. They have been found to be reliable markers for a variety of disease processes. In particular, a variety of miRNAs that are markers for neurodegenerative disease have been identified in cerebrospinal fluid. The aim of this study was to determine whether miRNAs could be identified in human perilymph potentially leading to the development of biomarkers for inner ear disease. Prospective sampling of human perilymph and its analysis were carried out. Patients undergoing surgery in which the inner ear is opened as part of the procedure (cochlear implantation, stapedectomy, labyrinthectomy) were recruited. A total of 2-5 μl of perilymph was collected and analyzed using Affymetrix GeneChip miRNA 4.0 microarrays. MiRNA common to all sampling approaches were selected. Analysis of miRNAs was carried out by evaluating miRNA targets in a cochlear transcriptome library using the Ingenuity Pathway Analysis software package. MiRNAs could be isolated from the perilymph of all patients. Evaluation of miRNAs shows the presence of miRNA populations that are predicted to interact with genes expressed in the inner ear. Additional analysis of miRNA populations shows that perilymph miRNAs could be linked to pathways associated with hearing disorders. Sampling of human perilymph is feasible and can potentially identify miRNAs associated with hearing disorders.

Abstract 3107: Modeling DICER1 syndrome in cells

Abstract DICER1 is an endoribonuclease central to generating microRNAs (miRNAs), small RNA molecules that downregulate the expression of approximately 30% of protein-coding genes. Germ-line mutations in DICER1 have been identified in patients afflicted with a pleiotropic tumor predisposition syndrome, usually referred to as DICER1 syndrome (OMIM 606241). DICER1 syndrome is thought to originate from a mesenchymal cell bearing a frameshift or nonsense mutation on one allele and a missense mutation affecting the RNase IIIb domain on the other allele. While interest in the role of DICER1 in normal development and pathogenesis has been explored in both the knockout and overexpression context, neither situation is relevant for understanding DICER1 syndrome. We hypothesize that defective DICER1 protein production in a mesenchymal stem cell at a critical time in development leads to altered miRNA populations that, in turn, initiate or prime cells for tumorigenesis. The goal of this study was to develop and characterize a model of the putative tumor-initiating cell namely a mesenchymal stromal cell (MSC) bearing a DICER1 RNase IIIb mutation. SV-40 immortalized mouse mesenchymal stromal cells (MSCs) containing a homozygous floxed exon for DICER1 were ex vivo cre inactivated to produce Dicer-/- MSCs. Into these Dicer-/- MSCs, a FLAG-tagged human DICER1, a FLAG-tagged RNase IIIa mutant DICER1, and a FLAG-tagged RNase IIIb mutant DICER1 were stably introduced. Cells were characterized by Western blot in response to serum starvation and stimulation. Their ability to form anchorage-independent colonies was assessed by soft agar assay pre- and post-oncogene introduction. Transcriptomes were interrogated by gene expression microarray. Results: Alterations in protein expression were observed in cells in response to starvation and stimulation. Certain cell lines were able to produce anchorage-independent colonies. Conclusions: We have created MSCs bearing RNase IIIb-mutated DICER1 to model DICER1 syndrome. We suggest that the ability for an MSC to produce only 3p miRNAs in the presence of an oncogene can allow for anchorage-independent growth. Citation Format: Mona K. Wu. Modeling DICER1 syndrome in cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3107.

Comprehensive analysis of blood cells and plasma identifies tissue-specific miRNAs as potential novel circulating biomarkers in cattle

BackgroundThe potential of circulating miRNAs as biomarkers of tissue function, both in health and disease, has been extensively demonstrated in humans. In addition, circulating miRNA biomarkers offer significant potential towards improving the productivity of livestock species, however, such potential has been hampered by the absence of information on the nature and source of circulating miRNA populations in these species. In addition, many miRNAs originally proposed as robust biomarkers of a particular tissue or disease in humans have been later shown not to be tissue specific and thus to actually have limited biomarker utility. In this study, we comprehensively analysed miRNA profiles in plasma and cell fractions of blood from cattle with the aim to identify tissue-derived miRNAs which may be useful as biomarkers of tissue function in this important food animal species.ResultsUsing small RNA sequencing, we identified 92 miRNAs with significantly higher expression in plasma compared to paired blood cell samples (n = 4 cows). Differences in miRNA levels between plasma and cell fractions were validated for eight out of 10 miRNAs using RT-qPCR (n = 10 cows). Among miRNAs found to be enriched in plasma, we confirmed miR-122 (liver), miR-133a (muscle) and miR-215 (intestine) to be tissue-enriched, as reported for other species. Profiling of additional miRNAs across different tissues identified the human homologue, miR-802, as highly enriched specifically in liver.ConclusionsThese results provide novel information on the source of bovine circulating miRNAs and could significantly facilitate the identification of production-relevant tissue biomarkers in livestock. In particular, miR-802, a circulating miRNA not previously identified in cattle, can reportedly regulate insulin sensitivity and lipid metabolism, and thus could potentially provide a specific biomarker of liver function, a key parameter in the context of post-partum negative energy balance in dairy cows.

MiRNA Long-Term Response to Early Metabolic Environmental Challenge in Hypothalamic Arcuate Nucleus.

Epidemiological reports and studies using rodent models indicate that early exposure to nutrient and/or hormonal challenges can reprogram metabolism at adulthood. Hypothalamic arcuate nucleus (ARC) integrates peripheral and central signals to adequately regulate energy homeostasis. microRNAs (miRNAs) participate in the control of gene expression of large regulatory networks including many signaling pathways involved in epigenetics regulations. Here, we have characterized and compared the miRNA population of ARC of adult male rats continuously exposed to a balanced metabolic environment to the one of adult male rats exposed to an unbalanced high-fat/high-carbohydrate/moderate-protein metabolic environment during the perinatal period and/or at adulthood that consequently displayed hyperinsulinemia and/or hyperleptinemia. We identified more than 400 miRNA species in ARC of adult male rats. By comparing the miRNA content of six biological replicates in each of the four perinatal/adult environments/rat groups, we identified the 10 miRNAs specified by clusters miR-96/182/183, miR-141/200c, and miR-200a/200b/429 as miRNAs of systematic and uncommonly high variation of expression. This uncommon variation of expression may underlie high individual differences in aging disease susceptibilities. By comparing the miRNA content of the adult ARC between the rat groups, we showed that the miRNA population was not affected by the unbalanced adult environment while, in contrast, the expression of 11 miRNAs was repeatedly impacted by the perinatal unbalanced environment. Our data revealed a miRNA response of adult ARC to early metabolic environmental challenge.

Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues.

RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues.

Recent Advances In Nutritional Sciences: An Overview Of Glycans And MiRNAs

There are many nutritional substances that humans consume on a daily basis: water, carbohydrates lipids and proteins are main biochemical components of food. But others in a smaller amount are vitamins minerals and enzymes. At a possibly lesser quantity are glycans and miRNAs. The presence of oligoglycans in all food sources has been an established fact for many years. These special carbohydrates are present as glycoconjugates (glycoproteins or glycolipids) in and on the surface of all the cells (glycocalyx) of all organisms that we eat and remain intact through the GI tract as we lack the enzymatic repertoire of the human body to unbind their particular β-linkages. Glycans bind to naturally present human lectins (through protein-carbohydrate interactions), but also with other human glycans (through carbohydrate-carbohydrate interactions, or CCI). Moreover, these glycans, like fibres, are digested by the gut microbiota that resides within the intestine. As our biochemistry shapes the composition of the microbiome, so does the composition of glycans and foods that we consume, triggering biological responses. miRNAs are small, single-stranded, 19- to 23- nucleotide-long RNA molecules and affect the stability of messenger RNA (mRNA) influencing protein synthesis. miRNAs are also present in foods and act on both the microbial composition in our gut and may be absorbed by the walls of the GI tract, demonstrating resistance to food processing and enzymatic attack. Though still a topic of controversy, these small, noncoding RNAs that control gene expression may directly enter into the circulating miRNA population of dietary exogenous miRNAs. It can hence be possible to identify a relationship between glycans and miRNAs in food on one side, microbiota composition on the other and the resultant health status of the host (immune system), on the third side.