Abundant miRNAs Research Articles

Deep RNA-Seq reveals miRNome differences in mammary tissue of lactating Holstein and Montb\xe9liarde cows

BackgroundGenetic polymorphisms are known to influence milk production and composition. However, the genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNAs) regulate gene expression. Previous research suggests that the high developmental potential of the mammary gland may depend in part on a specific miRNA expression pattern. The objective of the present study was to compare the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, which have different mammogenic potentials that are related to differences in dairy performance.ResultsMilk, fat, protein, and lactose yields were lower in Montbéliarde cows than in Holstein cows. We detected 754 distinct miRNAs in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows using RNA-Seq technology, among which 738 were known and 16 were predicted miRNAs. The 25 most abundant miRNAs accounted for 90.6% of the total reads. The comparison of their abundances in the mammary glands of Holstein versus Montbéliarde cows identified 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, and 2 (miR-100 and miR-146b) were highly expressed. Among the most abundant miRNAs, miR-186 is known to inhibit cell proliferation and epithelial-to-mesenchymal transition.Data mining showed that 17 differentially expressed miRNAs with more than 20 reads were involved in the regulation of mammary gland plasticity. Several of them may potentially target mRNAs involved in signaling pathways (such as mTOR) and lipid metabolism, thereby indicating that they could influence milk composition.ConclusionWe found differences in the mammary gland miRNomes of two dairy cattle breeds. These differences suggest a potential role for miRNAs in mammary gland plasticity and milk component synthesis, both of which are related to milk production and composition. Further research is warranted on the genetic regulation of miRNAs and their role in milk synthesis.

Variance component analysis of circulating miR-122 in serum from healthy human volunteers.

Micro-RNA (miR)-122 is a promising exploratory biomarker for detecting liver injury in preclinical and clinical studies. Elevations in serum or plasma have been associated with viral and autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma, and drug-induced liver injury (DILI). However, these associations were primarily based upon population differences between the disease state and the controls. Thus, little is known about the variability and subsequent variance components of circulating miR-122 in healthy humans, which has implications for the practical use of the biomarker clinically. To address this, we set out to perform variance components analysis of miR-122 in a cohort of 40 healthy volunteers. Employing a quantitative real-time polymerase chain reaction (qRT-PCR) assay to detect miR-122 and other circulating miRNAs in human serum, the relative expression of miR-122 was determined using two different normalization approaches: to the mean expression of a panel of several endogenous miRNAs identified using an adaptive algorithm (miRA-Norm) and to the expression of an exogenous miRNA control (Caenorhabditis elegans miR-39). Results from a longitudinal study in healthy volunteers (N = 40) demonstrated high variability with 117- and 111-fold 95% confidence reference interval, respectively. This high variability of miR-122 in serum appeared to be due in part to ethnicity, as 95% confidence reference intervals were approximately three-fold lower in volunteers that identified as Caucasian relative to those that identified as Non-Caucasian. Variance analysis revealed equivalent contributions of intra- and inter-donor variability to miR-122. Surprisingly, miR-122 exhibited the highest variability compared to other 36 abundant miRNAs in circulation; the next variable miRNA, miR-133a, demonstrated a 45- to 62-fold reference interval depending on normalization approaches. In contrast, alanine aminotransferase (ALT) activity levels in this population exhibited a 5-fold total variance, with 80% of this variance due to inter-donor sources. In conclusion, miR-122 demonstrated higher than expected variability in serum from healthy volunteers, which has implications for its potential utility as a prospective biomarker of liver damage or injury.

Differential microRNA Expression in Porcine Endometrium Involved in Remodeling and Angiogenesis That Contributes to Embryonic Implantation.

Background: In western swine breeds, up to 30% of embryonic losses occur during early pregnancy, and the majority of embryonic losses happens during implantation. In this period, maternal recognition of pregnancy begins to occur and blastocysts undergo dramatic morphologic changes. As with other species, changes in the uterine environment plays an important role in the process of embryo implantation in pigs. Erhualian (ER) pigs, one of the Chinese Taihu swine breeds, are known to have the highest litter size in the world. Experiments demonstrated that the greater embryonic survival on gestation day (GD) 12 in Chinese Taihu pigs is one important factor that contributes to enhanced litter size. This is largely controlled by maternal genes. In this study, endometrial samples were collected from pregnant Landrace×Large Yorkshire (LL) sows (parity 3) and ER sows (parity 3) on GD12 and the expression profiles of microRNAs (miRNAs) in the endometrium were compared between ER and LL using miRNA-seq technology. Results: A total of 288 miRNAs were identified in the pig endometrium, including 202 previously known and 86 novel miRNAs. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that highly abundant miRNAs might affect endometrial remodeling. Comparison between LL and ER sows revealed that 96 known miRNAs were differentially expressed between the two groups (including 78 up-regulated and 18 down-regulated miRNAs in ER compared to LL). Bioinformatics analysis showed that the target genes of some differentially expressed miRNAs were involved in pathways related to angiogenesis, proliferation, apoptosis, and tissue remodeling, which play critical roles in implantation by regulating endometrial structural changes and secretions of hormones, growth factors, and nutrients. Furthermore, the results demonstrated that insulin-like growth factor-1 protein expression was directly inhibited by miR-206. The lower expression of miR-206 in ER compared to LL might facilitate the angiogenesis of the endometrium during embryo implantation. Conclusions: The identified miRNAs that are differentially expressed in the endometrium of ER and LL pigs will contribute to the understanding of the role of miRNAs in embryonic implantation and the molecular mechanisms of the highest embryonic survival in Chinese ER pigs.

Endothelial progenitor cell-derived exosomes facilitate vascular endothelial cell repair through shuttling miR-21-5p to modulate Thrombospondin-1 expression.

Background: Our previous studies observed that administration of exosomes from endothelial progenitor cells (EPC) facilitated vascular repair in the rat model of balloon injury. However, the molecular events underlying this process remain elusive. Here, we aim to interrogate the key miRNAs within EPC-derived exosomes (EPC-exosomes) responsible for the activation of endothelial cell (EC) repair. Methods: The efficacy of EPC-exosomes in re-endothelialization was examined by Evans Blue dye and histological examination in the rat model of balloon-induced carotid artery injury. The effects of EPC-exosomes on human vascular EC (HUVEC) were also studied by evaluating the effects on growth, migratory and tube formation. To dissect the underlying mechanism, RNA-sequencing assays were performed to determine miRNA abundance in exosomes and mRNA profiles in exosome-treated HUVECs. Meanwhile, in vitro loss of function assays identified an exosomal miRNA and its target gene in EC, which engaged in EPC-exosomes-induced EC repair. Results: Administration of EPC-exosomes potentiated re-endothelialization in the early phase after endothelial damage in the rat carotid artery. The uptake of exogenous EPC-exosomes intensified HUVEC in proliferation rate, migration and tube-forming ability. Integrative analyses of miRNA-mRNA interactions revealed that miR-21-5p was highly enriched in EPC-exosomes and specifically suppressed the expression of an angiogenesis inhibitor Thrombospondin-1 (THBS1) in the recipient EC. The following functional studies demonstrated a fundamental role of miR-21-5p in the pro-angiogenic activities of EPC-exosomes. Conclusions: The present work highlights a critical event for the regulation of EC behavior by EPC-exosomes, which EPC-exosomes may deliver miR-21-5p and inhibit THBS1 expression to promote EC repair.

Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress

MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS). However, miRNAs have also been implicated in transcriptional gene regulation and alternative splicing, events that are restricted to the cell nucleus. Here we performed nuclear-cytoplasmic fractionation in a mouse endothelial cell line and characterized the localization of miRNAs in response to hypoxia using small RNA sequencing. A highly diverse population of abundant miRNA species was detected in the nucleus, of which the majority (56%) was found to be preferentially localized in one compartment or the other. Induction of hypoxia resulted in changes in miRNA levels in both nuclear and cytoplasmic compartments, with the majority of changes being restricted to one location and not the other. Notably, the classical hypoxamiR (miR-210-3p) was highly up-regulated in the nuclear compartment after hypoxic stimulus. These findings reveal a previously unappreciated level of molecular complexity in the physiological response occurring in ischemic tissue. Furthermore, widespread differential miRNA expression in the nucleus strongly suggests that these small RNAs are likely to perform extensive nuclear regulatory functions in the general case.

Expression levels of selected cytokines and microRNAs in response to vitamin D supplementation in ultra-marathon runners

Ultra-marathon (UM) running is an extreme endurance exercise. However, the mechanisms triggered with its practice remain unclear. While it is documented that strenuous physical activity activates immune responses and vitamin D plays a role in immune system suppression, data on the relationship between vitamin D status and cytokine profile in athletic populations are limited. To analyse the relative mRNA expression levels of selected pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, TNF-α), COX-2, vitamin D receptor and abundance of selected inflammatory microRNAs (Hsa-miR-21, -miR-146a, -miR-150, -miR-155, -miR-222, -miR-223) before and after a 100 km race in amateur runners in the presence or absence of vitamin D supplementation. Twenty runners aged 36–40years were divided into two groups: with and without vitamin D3 supplementation (10,000units daily). Blood samples were collected before and 12 h after the UM. The mRNA expression levels of selected cytokines, COX-2 and VDR in peripheral blood and abundance of serum exosomal miRNAs were investigated using q-RT-PCR. After UM, the significant up-regulation of TNF-α and hsa-miR-155 and down-regulation of IL-1β were observed in the group with vitamin D supplementation. In its absence, hsa-miR-155 and -miR-223 were significantly up-regulated. Additionally, a reverse correlation was observed between IL-6 expression level and abundance of hsa-miR-155 and -miR-223 in both groups. No statistical differences were noted when the other miRNAs and genes were examined in the groups and at the time points. The UM-induced mRNA expression pattern of pro-inflammatory cytokines could be influenced by vitamin D supplementation and/or miRNA.

Abstract 2743: Mesothelioma exosomes as potential new cancer therapy

Abstract Malignant mesothelioma (MM) is a highly invasive cancer arising on the mesothelial surface of organ cavities, predominantly the pleura, and is a direct result of asbestos exposure. Once diagnosed, the disease is virtually incurable with a median lifespan of 6-12 months. Additionally, there is no means of early diagnosis prior to disease symptoms, and the standard of care chemotherapeutics do not extend the life of patients. Taken together, these facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome contained microRNAs (miRNAs). Exosomes are vesicles secreted from every known mammalian cell type and present in essentially all body fluids. Most notably, exosomes play an outsize role in cancer biology. We initially sought to uncover the exosomal miRNAs secreted from MM tumor cells and found a unique signature associated with MM cancer exosomes compared to their mesothelial cell counterparts. The surprising discovery, however, was that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly the pro-apoptotic miR-16-5p. This led us to hypothesize that MM preferentially secreted tumor-suppressor miRNAs via exosomes in order to avoid their effect of killing the tumor cells. Through three novel avenues of potential therapeutic advance, coupled with the possibility of using these exosomal miRNAs as future biomarkers, we have embarked on an innovative strategy to kill MM tumor cells. We have employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16 exosome loss and rebuilding the cellular miR-16 leading to cell death, decreased migration/invasion, and loss of miR-16 target oncogenic proteins CCND1 and BCL2. In addition, we have force-fed concentrated MM tumor exosomes back to MM tumor cells, which led to remarkable levels of cell death, and a reduction in the same oncogenes, CCND1 and BCL2 as seen in our exosome inhibition trials. We have recapitulated these results with direct transfection of miR-16, leading to very comprehensive and convincing evidence that MM tumor cells do in fact secrete high levels of miR-16, and that when re-delivered back to the producer tumor cells through exosome inhibition, exosome force-feeding, or direct transfection, leads to cancer cell death and loss of oncogenic proteins CCND1 and BCL2. Our data, thus far, provide striking new information on a tumorigenic mechanism of MM tumor cells by preferential miR-16 secretion. Moreover we indicate possible miRNA biomarkers through exosomes, and show a never before proposed approach to targeting the death of MM cancer cells by blocking exosome secretion or force-feeding back their own exosomes. This study is supported majorly by Department of Defense, UVM REACH, the UVM Department of Pathology Fellowship, and in small part by NIH RO1. Citation Format: Phillip B. Munson, Elizabeth M. Hall, Arti Shukla. Mesothelioma exosomes as potential new cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2743.

Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis

Abstract Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis. Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2355.

Abstract 1793: Optimal quantification of a circulating miRNA profile predictive for treatment response with droplet digital PCR in patients with multi-organ metastatic colorectal cancer

Abstract Introduction: Presence of microRNAs (miRNAs) in blood specimens hold promise for a predictive biomarker for treatment response in clinical practice as they are relatively resistant to degradation. Previously, we identified a six miRNA expression signature from tumor tissue that predicted response to first line systemic treatment in patients with metastatic colorectal cancer (mCRC) when combined with clinicopathological factors. The six miRNA expression signature consists of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p. The aim of this study is to determine whether circulating levels of these miRNAs will be predictive for treatment outcome as well. Therefore we first studied whether these six miRNAs can be adequately measured in blood samples of patients with mCRC undergoing first line chemotherapy. Droplet digital PCR (ddPCR) technology was used for optimal quantification of miRNAs compared to normal quantitative PCR, because of low abundance of tumor specific miRNAs in blood. Methods: Serum samples of 120 patients with multi-organ mCRC who participated in a multi-center randomized controlled clinical trial were analyzed. RNA was isolated with miRNeasy Serum/Plasma Advanced Kit of Qiagen. Expression levels of the six miRNA expression signature was determined with ddPCR by using the miRCURY LNA miRNA PCR system combined with EvaGreen. Primer concentration and annealing temperature were first optimized per specific miRNA assay for ddPCR. RNA spike cel-miR-39-3p was used for quality control. Analysis was performed on samples with at least a minimal amount of 10.000 obtained droplets. Absolute quantification per patient is displayed as copies per µl (cop/µl) as defined by Quantasoft software. Results: In 120 blood samples miRNAs were measured by ddPCR. The most optimal primer concentrations for the different miRNAs was 0,5 µl for cel-miR-39-3p and 1,0 µl for the other 6 miRNAs. For cel-miR-39-3p, miR-17-5p, miR-20a-5p and miR-92a-3p the most efficient annealing temperature was 53 °C and for miR-30a-5p, miR-92b-3p and miR-98-5p this was 58 °C. The mean number of obtained droplets was 15.044 (SD 1922). The highest amount of detected copies were observed in miR-17-5p (644,4 cop/µl), miR-20a-5p (1037,1 cop/µl) and miR-92a-3p (1136,0 cop/µl). Lower expression levels were observed in miR-30a-5p (9,88 cop/µl), miR-92b-3p (1,6 cop/µl) and miR-98-5p (6,9 cop/µl). Conclusion: We demonstrated that with ddPCR absolute quantification of these six miRNAs in serum samples was efficient and reliable. Further analysis will reveal whether circulating miRNA expression levels can be used as a liquid biomarker for response to first line systemic treatment in patients with multi-organ mCRC. Citation Format: Lotte Bakkerus, Dennis Poel, Elske C. Gootjes, Cornelis Verhoef, Tineke E. Buffart, Hendrik M. Verheul. Optimal quantification of a circulating miRNA profile predictive for treatment response with droplet digital PCR in patients with multi-organ metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1793.

Strand displacement dual amplification miRNAs strategy with FRET between NaYF4:Yb,Tm/Er upconversion nanoparticles and Ti3C2 nanosheets

Due to the low abundance of miRNAs and the coordinated regulation of multiple miRNAs toward some biological processes, a method that can sensitively and simultaneously detect multiple miRNAs is needed. Here, a strand displacement dual amplification (SDDA) strategy was developed for multiple miRNAs analyses using a unique single strand-double strand-single strand DNA (sdsDNA), which generated by the target recognition probe hybridizing with the site region probe. During the process of SDDA, miRNA hybridized to the target recognition domain of sdsDNA, thereby releasing the site, which triggered the first strand displacement reaction. This strand displacement event resulted in the release of the other site, triggering the second strand displacement reaction. Both two strand displacement reactions in each assembly circle were integrated for amplified signals, which were tested by a fluorescence resonance energy transfer (FRET) assay. NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets were used as the energy donor-acceptor pair. By employing 1:2 ratio of donor-acceptor, the energy transfer efficiency between NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets could reach 90%, which enhanced signal-to-background ratio. Combining the benefits of dual amplification and efficient energy transfer on miRNAs detection, this assay exhibited a linear range from 5 fM to 100 pM for miRNA-21 and miRNA-10b. Moreover, this assay was applied to estimate the expression of miRNA-21 and miRNA-10b in human hepatoma and hepatocyte cell lines. Therefore, this FRET-based SDDA strategy has been used for highly sensitive detection of multiple miRNAs in cell lysate and facilitates the development of efficient biosensor platform.

Caloric Restriction Induces MicroRNAs to Improve Mitochondrial Proteostasis.

SummaryBoth caloric restriction (CR) and mitochondrial proteostasis are linked to longevity, but how CR maintains mitochondrial proteostasis in mammals remains elusive. MicroRNAs (miRNAs) are well known for gene silencing in cytoplasm and have recently been identified in mitochondria, but knowledge regarding their influence on mitochondrial function is limited. Here, we report that CR increases miRNAs, which are required for the CR-induced activation of mitochondrial translation, in mouse liver. The ablation of miR-122, the most abundant miRNA induced by CR, or the retardation of miRNA biogenesis via Drosha knockdown significantly reduces the CR-induced activation of mitochondrial translation. Importantly, CR-induced miRNAs cause the overproduction of mtDNA-encoded proteins, which induces the mitochondrial unfolded protein response (UPRmt), and consequently improves mitochondrial proteostasis and function. These findings establish a physiological role of miRNA-enhanced mitochondrial function during CR and reveal miRNAs as critical mediators of CR in inducing UPRmt to improve mitochondrial proteostasis.

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

MicroRNA Profiling of Exosomes Derived from Red Blood Cell Units: Implications in Transfusion-Related Immunomodulation.

Purpose To elucidate the microRNAs existent in exosomes derived from stored red blood cell (RBC) unit and their potential function. Materials and Methods Exosomes were isolated from the supernatant derived from stored RBC units by sequential centrifugation. Isolated exosomes were characterized by TEM (transmission electron microscopy), western blotting, and DLS (dynamic light scattering). MicroRNA (miRNA) microarray was performed to detect the expression of miRNAs in 3 exosome samples. Results revealed miRNAs that were simultaneously expressed in the 3 exosome samples and were previously reported to exist in mature RBCs. Functions and potential pathways of some detected miRNAs were illustrated by bioinformatic analysis. Validation of the top 3 abundant miRNAs was carried out by qRT-PCR (quantitative reverse transcription‐polymerase chain reaction). Results TEM and DLS revealed the mean size of the exosomes (RBC-derived) as 64.08 nm. These exosomes exhibited higher abundance of short RNA than the long RNA. 78 miRNAs were simultaneously detected in 3 exosome samples and mature RBCs. Several biological processes might be impacted by these miRNAs, through their target gene(s) enriched in a particular signalling pathway. The top 3 (abundant) miRNAs detected were as follows: miR-125b-5p, miR-4454, and miR-451a. qRT-PCR revealed higher abundance of miR-451a than others. Only miR-4454 and miR-451a abundance tended to increase with increasing storage time. Conclusion Exosomes derived from stored RBC units possessed multiple miRNAs and, hence, could serve various functions. The function of exosomes (RBC-derived) might be implemented partly by the predominantly enriched miR-451a.

Social status regulates the hepatic miRNAome in rainbow trout: Implications for posttranscriptional regulation of metabolic pathways.

Juvenile rainbow trout develop social hierarchies when held in dyads, and the development of socially subordinate (SS) and social dominance (SD) phenotypes in this context has been linked to specific changes in the hepatic energy metabolism of all major macronutrients. Following our recently reported finding that transcript abundance of drosha, a key component of the microRNA (miRNA) biogenesis pathway, is increased in paired juvenile rainbow trout irrespective of social status compared to socially isolated (SI) controls, we here determined global changes of the hepatic miRNA pathway genes in detail at the transcript and protein level. Both socially SD and SS rainbow trout exhibited increased Ago2 protein abundance compared to SI rainbow trout, suggesting that hepatic miRNA function is increased in rainbow trout maintained in dyads. Given the well-described differences in hepatic intermediary metabolism between socially SD and SS rainbow trout, and the important role of miRNAs in the posttranscriptional regulation of metabolic pathways, we also identified changes in hepatic miRNA abundance between socially SS and SD rainbow trout using small RNA next generation sequencing. We identified a total of 24 differentially regulated miRNAs, with 15 miRNAs that exhibited increased expression, and 9 miRNAs that exhibited decreased expression in the liver of socially SS trout compared to socially SD trout. To identify potential miRNA-dependent posttranscriptional regulatory pathways important for social status-dependent regulation of hepatic metabolism in rainbow trout, we used an in silico miRNA target prediction and pathway enrichment approach. We identified enrichment for pathways related to metabolism of carbohydrates, lipids and proteins in addition to organelle-specific processes involved in energy metabolism, especially mitochondrial fusion and fission. Select predicted miRNA-mRNA target pairs within these categories were quantitatively analyzed by real-time RT-PCR to validate candidates for future studies that will probe the functional metabolic roles of specific hepatic miRNAs in the development of socially SD and SS metabolic phenotypes.

3' Uridylation Confers miRNAs with Non-canonical Target Repertoires.

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3' uridylation. However, the ways in which thesesequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.

CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries.

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9–based Depletion of Abundant Species by Hybridization (‘DASH’) to smRNA-seq, which we have termed miRNA and Adapter Dimer—DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma

BackgroundCirculating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues.ResultsDifferent RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2–3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation.ConclusionsSmall RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.

In Situ Hybridization for Detecting Mature MicroRNAs In Vivo at Single-Cell Resolution.

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. © 2019 by John Wiley & Sons, Inc.

Altered microRNA expression profiles in large offspring syndrome and Beckwith-Wiedemann syndrome

ABSTRACTThe use of assisted reproductive technologies (ART) can induce a congenital overgrowth condition in humans and ruminants, namely Beckwith-Wiedemann syndrome (BWS) and large offspring syndrome (LOS), respectively. Shared phenotypes and epigenotypes have been found between BWS and LOS. We have observed global misregulation of transcripts in bovine foetuses with LOS. microRNAs (miRNAs) are important post-transcriptional gene expression regulators. We hypothesize that there is miRNA misregulation in LOS and that this misregulation is shared with BWS. In this study, small RNA sequencing was conducted to investigate miRNA expression profiles in bovine and human samples. We detected 407 abundant known miRNAs and predicted 196 putative miRNAs from the bovine sequencing results and identified 505 abundant miRNAs in human tongue. Differentially expressed miRNAs (DE-miRNAs) were identified between control and LOS groups in all tissues analysed as well as between BWS and control human samples. DE-miRNAs were detected from several miRNA clusters including DLK1-DIO3 genomic imprinted cluster in LOS and BWS. DNA hypermethylation was associated with downregulation of miRNAs in the DLK1-DIO3. mRNA targets of the DE-miRNAs were predicted and signalling pathways associated with control of organ size (including the Hippo signalling pathway), cell proliferation, apoptosis, cell survival, cell cycle, and cell adhesion were found to be enriched with these genes. Yes associated protein 1 (YAP1) is the core effector of the Hippo signalling pathway, and increased level of active (non-phosphorylated) YAP1 protein was detected in skeletal muscle of LOS foetuses. Overall, our data provide evidence of miRNA misregulation in LOS and BWS.

Involvement of hippocampal miR-664-3p in electroacupuncture induced improvement of learning-memory ability of cerebral ischemia-reperfusion rats

To explore the effect of electroacupuncture (EA) on learning-memory ability and its correlation with miR-664-3p levels in the hippocampus in cerebral ischemia-reperfusion injury (CI/RI) rats. A total of 27 male SD rats were randomly divided into sham operation, CI/RI model and EA groups (n＝9 in each group). The CI/RI model was established by occlusion of the middle cerebral artery (MACO) according to the modified Longa methods. EA was applied to "Baihui" (GV20) and "Shenting" (GV24) for 20 min, once a day for 7 days. The Morris water maze tests (place navigation tasks and spatial probe trials) were used to evaluate the rats' learning-memory ability. After EA intervention, the hippocampal tissue was collected to measure the mRNA and miRNA expression profiles by using high-throughput next generation sequencing. Bioinformatics analyses including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to screen the most abundant differentially-expressed miRNAs and mRNAs, particularly the target genes of miR-664-3p. Their biological functional categories including the key biochemical metabolic pathways and signaling pathways involved in CI and EA effect in the left hippocampus tissue were analyzed. The average escape latency of place navigation tests was significantly longer in the CI/RI model group than in the sham operation group (P<0.01), and obviously shorter in the EA group than in the model group (P<0.01); and the times of escape-platform quadrant crossing of the spatial probe trials were considerably fewer in the CI/RI model group than in the sham operation group (P<0.05), and markedly more in the CI/RI EA group than in the model group (P<0.05). Combined analysis of miRNA and mRNA showed that the differentially-expressed target genes of miR-664-3p were significantly up-regulated after CI/RI relevant to the sham operation rats (P<0.05) and notably down-regulated in the EA group relevant to the CI/RI model group (P<0.05). The GO analysis displayed that the target genes of miR-664-3p were significantly enriched in the category of cell transportation activity, ion binding，and transmembrane transporter activity, the production of precursor metabolites and energy, transportation, signal transduction，localization, etc. KEGG analysis showed that the target genes of miR-664-3p in this study were mainly enriched in the thyroid hormone pathway, Hippo signaling pathway, MAPK signal transduction pathway，cAMP signaling pathway, neurotrophic factor signal transduction pathway, etc. EA of GV20 and GV24 can improve the learning-memory ability of CI/RI rats, which is closely related to its effect in down-regulating the levels of hippocampal target genes of miR-664-3p involving cellular transmembrane transportation，cell to environment interaction，cell-cell communication, etc.