Authors

RNA quality control in miRNA expression analysis Application Not

Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs

Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments

Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves

Prostaglandins in Superovulation Induced Bovine Follicles During the Preovulatory Period and Early Corpus Luteum

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation

Tumor Necrosis Factor Alpha and Insulin-Like Growth Factor 1 Induced Modifications of the Gene Expression Kinetics of Differentiating Skeletal Muscle Cells

Introduction TNF-alpha levels are increased during muscle wasting and chronic muscle degeneration and regeneration processes, which are characteristic for primary muscle disorders. Pathologically increased TNF-alpha levels have a negative effect on muscle cell differentiation efficiency, while IGF1 can have a positive effect;therefore, we intended to elucidate the impact of TNF-alpha and IGF1 on gene expression during the early stages of skeletal muscle cell differentiation. Methodology/Principal Findings This study presents gene expression data of the murine skeletal muscle cells PMI28 during myogenic differentiation or differentiation with TNF-alpha or IGF1 exposure at 0 h, 4 h, 12 h, 24 h, and 72 h after induction. Our study detected significant coregulation of gene sets involved in myoblast differentiation or in the response to TNF-alpha. Gene expression data revealed a time-and treatment-dependent regulation of signaling pathways, which are prominent in myogenic differentiation. We identified enrichment of pathways, which have not been specifically linked to myoblast differentiation such as doublecortin-like kinase pathway associations as well as enrichment of specific semaphorin isoforms. Moreover to the best of our knowledge, this is the first description of a specific inverse regulation of the following genes in myoblast differentiation and response to TNF-alpha: Aknad1, Cmbl, Sepp1, Ndst4, Tecrl, Unc13c, Spats2l, Lix1, Csdc2, Cpa1, Parm1, Serpinb2, Aspn, Fibin, Slc40a1, Nrk, and Mybpc1. We identified a gene subset (Nfkbia, Nfkb2, Mmp9, Mef2c, Gpx, and Pgam2),which is robustly regulated by TNF-alpha across independent myogenic differentiation studies. Conclusions This is the largest dataset revealing the impact of TNF-alpha or IGF1 treatment on gene expression kinetics of early in vitro skeletal myoblast differentiation. We identified novel mRNAs, which have not yet been associated with skeletal muscle differentiation or response to TNF-alpha. Results of this study may facilitate the understanding of transcriptomic networks underlying inhibited muscle differentiation in inflammatory diseases

Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow

Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will en-courage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions

Detection of Atherosclerosis by Small RNA-Sequencing Analysis of Extracellular Vesicle Enriched Serum Samples

Atherosclerosis can occur throughout the arterial vascular system and lead to various diseases. Early diagnosis of atherosclerotic processes and of individual disease patterns would be more likely to be successful if targeted therapies were available. For this, it is important to find reliable biomarkers that are easily accessible and with little inconvenience for patients. There are many cell culture, animal model or tissue studies that found biomarkers at the microRNA (miRNA) and mRNA level describing atherosclerotic processes. However, little is known about their potential as circulating and liquid biopsy markers in patients. In this study, we examined serum-derived miRNA – profiles from 129 patients and 28 volunteers to identify potential biomarkers. The patients had four different atherosclerotic manifestations: abdominal aneurysm (n = 35), coronary heart disease (n = 34), carotid artery stenosis (n = 24) and peripheral arterial disease (n = 36). The samples were processed with an extracellular vesicle enrichment protocol, total-RNA extraction and small RNA-sequencing were performed. A differential expression analysis was performed bioinformatically to find potentially regulated miRNA biomarkers. Resulting miRNA candidates served as a starting point for an overrepresentation analysis in which relevant target mRNAs were identified. The Gene Ontology database revealed relevant biological functions in relation to atherosclerotic processes. In patients, expression of specific miRNAs changed significantly compared to healthy volunteers; 27 differentially expressed miRNAs were identified. We were able to detect a group-specific miRNA fingerprint: miR-122-5p, miR-2110 and miR-483-5p for abdominal aortic aneurysm, miR-370-3p and miR-409-3p for coronary heart disease, miR-335-3p, miR-381-3p, miR493-5p and miR654-3p for carotid artery stenosis, miR-199a-5p, miR-215-5p, miR-3168, miR-582-3p and miR-769-5p for peripheral arterial disease. The results of the study show that some of the identified miRNAs have already been associated with atherosclerosis in previous studies. Overrepresentation analysis on this data detected biological processes that are clearly relevant for atherosclerosis, its development and progression showing the potential of these miRNAs as biomarker candidates. In a next step, the relevance of these findings on the mRNA level is to be investigated and substantiated

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development

Background: miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. Methods: NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based profiling. Significant candidates were confirmed in EVs isolated by size-exclusion chromatography from nasal lavages of children with mild-to-moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). Results: NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210, and miR-34a significantly correlated with lung function parameters in children (FEV1FVC%pred and FEF25-75%pred), thus lower sEV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. Conclusion: Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b, and miR-210, might be involved in the early development of a Th2 response in the airways and asthma

Extracellular Vesicle Associated miRNAs Regulate Signaling Pathways Involved in COVID-19 Pneumonia and the Progression to Severe Acute Respiratory Corona Virus-2 Syndrome

Background: Extracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19. Methods: We prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS. Results: DGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted<0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, padjusted=0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p. Conclusion: EV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets