The developmentally-timed decay of an essential microRNA family is seed sequence-dependent

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here we show that the mir-35 miRNA family undergoes regulated decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and sufficient for this regulation. Sequences outside the seed (3′ end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3′ end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3′ end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family′s target repertoire.

MicroRNA-183-3p Is a Predictor of Worsening Heart Failure in Adult Patients With Transposition of the Great Arteries and a Systemic Right Ventricle

Aim: MicroRNAs (miRNAs) have been shown to play an important role in the progression of heart failure (HF). The aim of our study was to analyze miRNAs in the blood of patients with transposition of the great arteries and a systemic right ventricle (TGA-RV) in order to identify those that predict worsening HF.Materials and Methods: In 36 patients with TGA-RV, SurePrint™ 8 × 60K Human v21 miRNA microarrays were used to determine the miRNA abundance profiles and compared to 35 age- and gender-matched healthy volunteers (HVs). MiRNAs that were most significantly abundant or best related to worsening HF were further validated by RT-qPCR.Results: Using miRNA array analysis, a total of 50 down-regulated and 56 up-regulated miRNAs were found to be differentially abundant in TGA-RV patients compared to HVs. Six of these 106 miRNAs were significantly related to worsening HF. After validation by RT-qPCR, four miRNAs turned out to be significantly associated with worsening HF, namely miR-150-5p, miR-1255b-5p, miR-423-3p, and miR-183-3p. In the stepwise multivariable Cox regression analysis, ejection fraction of the systemic RV, high sensitive TNT and miR-183-3p were found to be independent predictors of worsening HF (P = 0.001, P = 0.002, and P = 0.001, respectively).Conclusions: In patients with TGA-RV, miR-183-3p is an independent predictor of worsening HF and thus may be used as additional biomarker in the risk assessment of these patients.

Characterization of micro-RNA in women with different ovarian reserve

Women undergoing infertility treatment are routinely subjected to one or more tests of ovarian reserve. Therefore, an adequate assessment of the ovarian reserve is necessary for the treatment. In this study, we aimed to characterize the potential role of microRNAs (miRNAs) as biomarkers for women with different ovarian reserves. A total of 159 women were recruited in the study and classified according to their anti-Müllerian hormone (AMH) level into three groups: (1) low ovarian reserve (LAMH, n = 39), (2) normal ovarian reserve (NAMH, n = 80), and (3) high ovarian reserve (HAMH, n = 40). SurePrint Human miRNA array screening and reverse transcription-quantitative PCR (RT-qPCR) were respectively employed to screen and validate the miRNA abundance level in the three tested groups. Compared with NAMH, the abundance level of 34 and 98 miRNAs was found to be significantly altered in LAMH and HAMH, respectively. The abundance level of miRNAs was further validated by RT-qPCR in both, the screening samples as well as in an independent set of validation samples. The abundance levels of the validated miRNAs were significantly correlated with the AMH level. The best AUC value for the prediction of the increase and decrease in the AMH level was obtained for the miR-100-5p and miR-21-5p, respectively. The level of miRNAs abundance correlates with the level of AMH, which may serve as a tool for identifying women with a different ovarian reserve and may help to lay the ground for the development of novel diagnostic approaches.

Distribution of microRNAs associated with major depressive disorder among blood compartments

Objective Major depressive disorder (MDD) is a recurrent disorder with an increasing incidence. Alterations in key signaling pathways of the nervous system, such as the Wnt and MAPK pathways, mediated through microRNAs (miRNAs) provide crucial information regarding the etiopathology of MDD. We aimed to analyze whether the heterogeneity of literature findings regarding differential expression of miRNAs in the blood could arise from their different distributions among blood compartments. Methods We performed a pilot study analyzing the differential expression of miR-26a, miR-494, miR-30c, miR-93, and miR-101 and investigated their levels in white blood cells, total plasma (TP), exosomes from plasma, and exosome depleted plasma (EDP) in patients with MDD before and after antidepressant treatment with escitalopram and in healthy controls. Results MiR-494 was more abundant in EDP, and miR-26a and miR-30c were predominantly more abundant in TP relative to other blood compartments. Moreover, miR-30c, miR-101, and miR-26a, were significantly downregulated in TP of patients with MDD compared with controls. After antidepressant treatment, only miR-494 was significantly differently expressed in EDP. Conclusions This proof-of-principle study suggests that identifying the miRNA abundance in different blood compartments is crucial for biomarker development and could enrich the current knowledge regarding MDD pathophysiology.

Integrative Analysis of Gene Expression and miRNAs Reveal Biological Pathways Associated with Bud Paradormancy and Endodormancy in Grapevine

Transition of grapevine buds from paradormancy to endodormancy is coordinated by changes in gene expression, phytohormones, transcription factors, and other molecular regulators, but the mechanisms involved in transcriptional and post-transcriptional regulation of dormancy stages are not well delineated. To identify potential regulatory targets, an integrative analysis of differential gene expression profiles and their inverse relationships with miRNA abundance was performed in paradormant (long day (LD) 15 h) or endodormant (short day (SD), 13 h) Vitis riparia buds. There were 400 up- and 936 downregulated differentially expressed genes in SD relative to LD budsGene set and gene ontology enrichment analysis indicated that hormone signaling and cell cycling genes were downregulated in SD relative to LD buds. miRNA abundance and inverse expression analyses of miRNA target genes indicated increased abundance of miRNAs that negatively regulate genes involved with cell cycle and meristem development in endodormant buds and miRNAs targeting starch metabolism related genes in paradormant buds. Analysis of interactions between abundant miRNAs and transcription factors identified a network with coinciding regulation of cell cycle and epigenetic regulation related genes in SD buds. This network provides evidence for cross regulation occurring between miRNA and transcription factors both upstream and downstream of MYB3R1.

Platelet Activation Is Not Always Associated With Platelet-Related Plasma microRNA Abundance – Results From a Randomized Controlled Trial of Periodontal Patients

Platelets are involved in a variety of diseases, making their adequate functional assessment is essential. However, due to their easily activatable nature this has some methodological pitfalls. Therefore, the availability of stable, easily measurable surrogate markers would be beneficial. In this regard, some evidence suggests that certain microRNAs (miRNAs) circulating in plasma might be useful. We aimed to corroborate their suitability by analyzing plasma samples obtained in a randomized controlled trial, which assessed the effects of periodontal treatment on platelet function. We hypothesized that miRNA levels mirror changes of platelet activation and -function. Both platelet function and miRNA abundance were quantified using state-of-the-art flow cytometry and qPCR methods. The following miRNAs were quantified: 223-3p, 150-5p, 197-3p, 23a-3p, 126-3p, 24-3p, 21-5p, 27b-3p, 33a-5p, 320a, 191-5p, 28-3p, 451a, 29b-3p, and 1-3p. However, periodontal treatment did not affect the abundance of any investigated miRNAs to a relevant extent. Platelet activation and reactivity indices did neither correlate with any tested miRNA at baseline, nor after the treatment period. In addition, there was no evidence that investigated miRNAs were released by platelets, as suggested previously. In conclusion, our data suggest that in patients suffering from periodontal disease the investigated miRNAs are unlikely to be suitable biomarkers for platelet function. Our data aim to raise awareness that previously determined platelet activation dependent circulating miRNAs are not suitable as platelet biomarkers in all cohorts.

MiR-26a-5p as a Reference to Normalize MicroRNA qRT-PCR Levels in Plasma Exosomes of Pediatric Hematological Malignancies

Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.

Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles

The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material.

MicroRNA dilution during oocyte growth disables the microRNA pathway in mammalian oocytes

MicroRNAs (miRNAs) are ubiquitous small RNAs guiding post-transcriptional gene repression in countless biological processes. However, the miRNA pathway in mouse oocytes appears inactive and dispensable for development. We propose that marginalization of the miRNA pathway activity stems from the constraints and adaptations of RNA metabolism elicited by the diluting effects of oocyte growth. We report that miRNAs do not accumulate like mRNAs during the oocyte growth because miRNA turnover has not adapted to it. The most abundant miRNAs total tens of thousands of molecules in growing (∅ 40 μm) and fully grown (∅ 80 μm) oocytes, a number similar to that observed in much smaller fibroblasts. The lack of miRNA accumulation results in a 100-fold lower miRNA concentration in fully grown oocytes than in somatic cells. This brings a knock-down-like effect, where diluted miRNAs engage targets but are not abundant enough for significant repression. Low-miRNA concentrations were observed in rat, hamster, porcine and bovine oocytes, arguing that miRNA inactivity is not mouse-specific but a common mammalian oocyte feature. Injection of 250,000 miRNA molecules was sufficient to restore reporter repression in mouse and porcine oocytes, suggesting that miRNA inactivity comes from low-miRNA abundance and not from some suppressor of the pathway.