Whole Blood Holding Time Prior to Plasma Processing Alters microRNA Expression Profile

MicroRNAs (miRNAs) can exhibit aberrant expression under different physiological and pathological conditions. Therefore, differentially expressed circulating miRNAs have been a focus of biomarker discovery research. However, the use of circulating miRNAs comes with challenges which may hinder the reliability for their clinical application. These include varied sample collection protocols, storage times/conditions, sample processing and analysis methods. This study focused on examining the effect of whole blood holding time on the stability of plasma miRNA expression profiles. Whole blood samples were collected from healthy pregnant women and were held at 4°C for 30 min, 2 h, 6 h or 24 h prior to processing for plasma isolation. Plasma RNA was extracted and the expression of 179 miRNAs were analyzed. Unsupervised principal component analysis demonstrated that whole blood holding time was a major source of variation in miRNA expression profiles with 53 of 179 miRNAs showing significant changes in expression. Levels of specific miRNAs previously reported to be associated with pregnancy-associated complications such as hsa-miR-150-5p, hsa-miR-191-5p, and hsa-miR-29a-3p, as well as commonly used endogenous miRNA controls, hsa-miR-16-5p, hsa-miR-25-3p, and hsa-miR-223-3p were significantly altered with increase in blood holding time. Current protocols for plasma-based miRNA profiling for diagnostics describe major differences in whole blood holding periods ranging from immediately after collection to 26 h after. Our results demonstrate holding time can have dramatic effects on analytical reliability and reproducibility. This highlights the importance of standardization of blood holding time prior to processing for plasma in order to minimize introduction of non-biological variance in miRNA profiles.

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously non-annotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the 'Brain miRTExplorer' web application freely accessible for the community.

Mechanistic Basis of Arsenic Induced Carcinogenesis: Differential miRNA Expression

Arsenic, a toxic metalloid, provokes many detrimental consequences to human health. It is prevalent in earth's crust and poses a major threat to humans globally. Inorganic arsenic exposure occurs mainly via drinking water or food and is metabolized in mammals to form organic metabolites/ end products. Chronic exposure to arsenic causes lung, skin and urinary bladder cancers and increases the risks of liver, kidney and prostate cancers. Arsenic-induced ROS generation, disturbances in several signaling pathways, DNA repair inhibition, chromosomal aberrations, and epigenetic changes including alterations in DNA methylation, histone modifications and differential miRNA expression profiles are involved in cancer progression, and malignant transformation. However, details of arsenic-induced carcinogenesis and molecular mechanisms involved are still remaining obscure. MicroRNAs are post-transcriptional gene expression regulators and themselves may act as oncogenes and tumor suppressor genes. Differential miRNA expression is implicated in several human cancers. This review covers general mechanistic basis of arsenic-induced carcinogenesis, explores recent in-vitro, in-vivo and cohort studies on differential miRNA expression profiles and shares associated molecular mechanistic data on miRNA dysregulation and their functional consequences leading to arsenic induced tumorigenesis, metastasis and cancer, also discusses the future directions.

microRNA Profile Associated with Positive Lymph Node Metastasis in Early-Stage Cervical Cancer

Lymph node metastasis (LNM) is an important prognostic factor in cervical cancer (CC). In early stages, the risk of LNM is approximately 3.7 to 21.7%, and the 5-year overall survival decreases from 80% to 53% when metastatic disease is identified in the lymph nodes. Few reports have analyzed the relationship between miRNA expression and the presence of LNM. The aim of this study was to identify a subset of miRNAs related to LNM in early-stage CC patients. Formalin-fixed paraffin-embedded tissue blocks were collected from patients with early-stage CC treated by radical hysterectomy with lymphadenectomy. We analyzed samples from two groups of patients—one group with LNM and the other without LNM. Global miRNA expression was identified by microarray analysis, and cluster analysis was used to determine a subset of miRNAs associated with LNM. Microarray expression profiling identified a subset of 36 differentially expressed miRNAs in the two groups (fold change (FC) ≥ 1.5 and p < 0.01). We validated the expression of seven miRNAs; miR-487b, miR-29b-2-5p, and miR-195 were underexpressed, and miR-92b-5p, miR-483-5p, miR-4534, and miR-548ac were overexpressed according to the microarray experiments. This signature exhibited prognostic value for identifying early-stage CC patients with LNM. These findings may help detect LNM that cannot be observed in imaging studies.

PM10 Alters Trophoblast Cell Function and Modulates miR-125b-5p Expression

Air pollution is one of the largest global environmental health hazards that threaten premature mortality or morbidity. Particulate matter 10 (PM10) has been demonstrated to contribute to several human diseases via dysregulated miRNA expression. Trophoblast cells play a key role in implantation and placentation for a successful pregnancy. Nonetheless, the PM10 associated trophoblast cell functions during pregnancy and miRNA expression are still unknown. Our study showed that PM10 affected HTR-8/SVneo cell viability and also decreased cell proliferation, migration, and invasion. A high concentration of PM10 caused an increase in HTR-8/SVneo cell apoptosis. Treatment with PM10 induced inflammation through the upregulated IL-1β, IL-6, and TNF-α expression in trophoblast cells. In PM10-treated HTR-8/SVneo cells, miR-125b-5p expression was considerably increased and TXNRD1 was found to be negatively related to miR-125b-5p. Collectively, our findings revealed that PM10 could alter miR-125b-5p expression by targeting TXNRD1 and suppressing trophoblast cell functions. Additional investigations relating to the function of miR-125b-5p and its target on particulate pollution exposure in trophoblast are warranted for future biomarker or effective therapeutic approaches.

Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer

In complex biochemical systems, an enzyme, protein, or RNA, symbolized as E, has hundreds or thousands of substrates or interacting partners. The relative specificity hypothesis proposes that such an E would differentially interact with and influence its many distinct, downstream substrates, thereby regulating the underlying biological process (es). The importance of relative specificity has been underappreciated, and evidence of its physiological consequences particularly lacking. Previously we showed that human Drosha and Dicer ribonucleases (RNases) both discriminate their respective microRNA (miRNA) substrates, and that differential cleavage by Drosha contributes to global differential miRNA expression. If relative specificity is an important biological mechanism, it should be evolutionarily conserved. To test this hypothesis, we hereby examined the cleavage of hundreds of zebrafish and fruitfly miRNA intermediates by Drosha and Dicer and the impact on miRNA biogenesis in these organisms. We showed that Drosha action regulates differential miRNA expression in zebrafish and fruitflies and identified the conserved secondary structure features and sequences in miRNA transcripts that control Drosha activity and miRNA expression. Our results established the conservation of miRNA processing mechanisms and regulatory functions by Drosha and Dicer, greatly strengthened the evidence for the physiological consequences of relative specificity as well as demonstrated its evolutionary significance.

MicroRNA in refined diagnosis of choroidal melanoma

Epigenetic studies of the level of microRNAs in human oncogenesis indicate their signifi cant role in the development and growth of malignant tumors of various origins. The fi rst works on the role of microRNAs in patients with uveal melanoma appeared in 2008.The aim: to analyze the expression level of miRNA-126 and miRNA-223 in the plasma blood of patients and to determine their signifi cance in the refi ned diagnosis of choroidal melanoma. Materials and methods. We examined 84 patients with choroidal melanoma (CM), mean age – 63.4 ± 1.2 (35–86 y.o.). Localization – a single CM node with a thickness of 0.77–17.19 mm. The control group consisted of 28 volunteers, age – 62.9 ± 1.42 (45–78 y.o.). Plasma miRNA expression levels were determined by real-time PCR.Results. An increase in the level of expression of miRNA-223 and miRNA-126 in blood plasma was confi rmed in all 84 patients with choroidal melanoma N0M0 compared with the control group. An increase in the expression of miRNA-223 and miRNA-126 was proved with an increase in tumor prominence.Conclusion. The obtained results of an increase in the expression of miRNA-223 indicate an increase in cell proliferation, and an increase in the expression of miRNA-126 on the activation of angiogenesis in a growing tumor, which makes it possible to recommend a study of the level of miRNA-223 and miRNA-126 for a more accurate diagnosis of small CM in cases of difficulty of differential diagnosis with other tumor-like diseases of the choroid.

Orange Juice Attenuates Circulating miR-150-5p, miR-25-3p, and miR-451a in Healthy Smokers: A Randomized Crossover Study

Introduction: Tobacco smoke is associated with oxidative and inflammatory pathways, increasing the risk of chronic-degenerative diseases. Our goal was to evaluate the effects of acute “Pera” and “Moro” orange juice consumption on inflammatory processes and oxidative stress in microRNA (miRNA) expression in plasma from healthy smokers.Methods: This was a randomized crossover study that included healthy smokers over 18 years old. Blood samples were collected before and 11 h after beverage ingestion. Participants were instructed to drink 400 mL of Pera orange juice (Citrus sinensis), Moro orange juice (Citrus sinensis L. Osbeck), or water. Each subject drank the beverages in a 3-way crossover study design. Inflammatory and oxidative stress biomarkers and circulating miRNA expression profiles were determined. The subjects maintained their usual tobacco exposure during the experiment.Results: We included 18 individuals (12 men and 6 women), with 37.0 ± 12.0 years old. All subjects received the 3 interventions. Increased expression of circulating miRNAs (miR-150-5p, miR-25-3p, and miR-451a) was verified after cigarette smoking, which were attenuated after intake of both types of orange juice. There was no difference regarding serum levels of TNF-α, IL-6, MMP-9, and C-reactive protein. Despite the increased activity of serum superoxide dismutase and glutathione peroxidase after “Pera” or “Moro” orange juice intake, respectively, no changes in lipid hydroperoxide levels were detected.Conclusion: Tobaccos smokers showed increased expression of miR-150-5p, miR-25-3p, and miR-451a was noted, and attenuated by orange juice intake. miRNAs were predicted to regulate 244 target genes with roles in oxidative stress, PI3K-Akt, and MAPK signaling, which are pathways frequently involved in smoking-related cardiovascular diseases and cancer.

Renal Tissue miRNA Expression Profiles in ANCA-Associated Vasculitis—A Comparative Analysis

Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) comprises autoimmune disease entities that cause target organ damage due to relapsing-remitting small vessel necrotizing vasculitis, and which affects various vascular beds. The pathogenesis of AAV is incompletely understood, which translates to considerable disease- and treatment-related morbidity and mortality. Recent advances have implicated microRNAs (miRNAs) in AAV; however, their accurate characterization in renal tissue is lacking. The goal of this study was to identify the intrarenal miRNA expression profile in AAV relative to healthy, non-inflammatory and inflammatory controls to identify candidate-specific miRNAs. Formalin-fixed, paraffin-embedded renal biopsy tissue samples from 85 patients were obtained. Comprehensive miRNA expression profiles were performed using panels with 752 miRNAs and revealed 17 miRNA that differentiated AAV from both controls. Identified miRNAs were annotated to characterize their involvement in pathways and to define their targets. A considerable subset of differentially expressed miRNAs was related to macrophage and lymphocyte polarization and cytokines previously deemed important in AAV pathogenesis, lending credence to the obtained results. Interestingly, several members of the miR-30 family were detected. However, a validation study of these differentially expressed miRNAs in an independent, larger sample cohort is needed to establish their potential diagnostic utility.