miRNA Concentrations Research Articles

Duplex-Specific Nuclease-Assisted CRISPR-Cas12a Strategy for MicroRNA Detection Using a Personal Glucose Meter.

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. Nevertheless, the current CRISPR-Cas12a-based detection strategies always need bulky instruments or auxiliary devices to obtain a quantitative signal output, which restrains its point-of-care testing application. Herein, we proposed a duplex-specific nuclease-assisted CRISPR-Cas12a strategy to detect microRNA (miRNA) with a personal glucose meter. The target miRNA was first converted into an amplified initiator DNA via duplex-specific nuclease. Afterward, the initiator DNA activated the collateral cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA-modified magnetic beads, which led to the release of sucrase. The released sucrase was collected and then utilized to catalyze sucrose to glucose, which could be quantitatively detected by a personal glucose meter. The change in the glucose signal directly reflected the concentration of miRNA, which avoided expensive equipment for signal quantification. Two different miRNAs (miRNA21 and miRNA205) could be detected by simply changing the sequence of the template strand (H strand). The developed strategy showed high sensitivity with a limit of detection (LOD) of 2.4 and 1.1 pM for miRNA21 and miRNA205, respectively. In addition, good selectivity and anti-interference ability were achieved using this method, which enabled it promising for miRNA detection at the point-of-care.

Effects of silybin supplementation on nutrient digestibility, hematological parameters, liver function indices, and liver-specific mi-RNA concentration in dogs

BackgroundHepatopathies are an important group of disorders in dogs where proper nutritional care is crucial. Supplementation with a hepatoprotectant like silybin can improve liver function and should not interfere with nutrient digestibility. The purpose of this study was to investigate the effect of both pure silybin and commercial hepatoprotectant on nutrients digestibility, liver function indices and health status in healthy dogs (EXP1). Moreover, the second experiment (EXP2) investigated the effect of commercial hepatoprotectant on liver function tests and liver-associated miRNAs concentration in dogs with idiopathic liver disorder.ResultsNutrient digestibility was not affected by treatment in EXP1. Supplementation did alter the serum fatty acid profile, with no clinical relevance. The levels of liver markers such as ALT, AST and GGT significantly decreased. In EXP2, supplementation with commercial hepatoprotectant containing silybin improved liver function tests. A decrease was observed in liver serum markers such as ALT, AST and miR122 concentration.ConclusionsEXP1 confirmed that silybin (whether pure or as a commercial hepatoprotectant) does not interfere with digestion which subsequently exerts no detrimental effect on dogs’ health and metabolism. In EXP2, dietary supplementation with commercial hepatoprotectant containing silybin resulted in a decreased activity of serum liver markers, accompanied by a decrease in the concentration of liver-specific miRNA molecules. Liver function indices were consequently improved. Silybin supplementation can thus serve as an effective therapeutical tool in dogs with hepatopathies.

In Situ Formation of Ag2S Nanoparticles on a Paper‐Supported Wettable Microchip for High‐Throughput Photothermal Sensing of MicroRNA

AbstractPhotothermal biosensors have emerged as an attractive strategy for point‐of‐care testing (POCT). However, developing a photothermal biosensor with good accuracy to realize high‐throughput detection is still a big challenge. Herein, a photothermal biosensor comprised of a paper‐supported wettable microchip was proposed for sensitive, accuracy, high‐throughput detection of microRNA (miRNA). Hybridization chain reaction (HCR) products on the wettable paper induced by target miRNA were used as a template for in situ formation of Ag2S nanoparticles (NPs), which can translate miRNA concentration into temperature change. The developed photothermal biosensor with low sample volume exhibited high sensitivity with a detection limit of 49.7 pM. In addition, the biosensor showed excellent reproducibility and selectivity, and the miRNA content in cell lysates was successfully assayed. The proposed photothermal biosensor shows great potential in biomedical application and clinical diagnosis.

MO448MICRORNAS IMPLICATED IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims The gene program is controlled at the post-transcriptional level by the action of small non-coding RNAs known as microRNAs (miRNAs), short, single-stranded molecules that control mRNA stability or translational repression via base pairing with regions in the 3' untranslated region of their target mRNAs. Recently, considerable progress has been made to elucidate the roles of miRNAs in vascular pathogenesis and develop the use of miRNAs as biomarkers, and innovative drugs. We demonstrated during the last decade that miRNAs miR-126 and miR-223 are implicated in the course of chronic kidney disease (CKD) and cardiovascular damage. miR-223 expression is enhanced in vascular smooth muscle cells (VSMCs) subjected to an uremic toxin and also in aortas of a murine model of CKD. As restenosis is a common complication of angioplasty, in which neointimal hyperplasia results from migration of VSMCs into the vessel lumen we measured the effect of miR-223 modulation on restenosis in a rat model of carotid artery after balloon injury. We over-expressed and inhibited miR-223 expression using adenoviral vectors, coding a pre-miR-223 sequence or a sponge sequence, used to trap endogenous microRNA, respectively. We demonstrated that inhibiting miR-223 function significantly reduced neointimal hyperplasia by almost half in carotids. Thus down-regulating miR-223 could be a potential therapeutic approach to prevent restenosis after angioplasty. We also correlated miR-126 and miR-223 expression with clinical outcomes in a large cohort of CKD patients, in collaboration with the University Hospital of Ghent (Belgium) and Ambroise Paré Hospital, France. We evaluated both miRNA’s link with all-cause mortality and cardiovascular and renal events over a 6-year follow-up period. The serum levels of miR-126 and miR-223 were decreased as CKD stage advanced, and patients with higher levels of miR-126 and miR-223 had a higher survival rate. Similar results were observed for cardiovascular and renal events. In conclusion, CKD is associated with a decrease in circulating miR-126 and miR-223 levels in CKD patients. We will also present links between several uremic toxin concentrations and miRNA concentration in the patients of this cohort. Finally, anemia is a common feature of CKD that is associated with cardiovascular disease and poor clinical outcomes. A mixture of uremic toxins accumulates in the blood of CKD patients during the course of the disease, and there is good evidence that they modulate erythropoiesis, explaining at least partly anemia. The exact molecular mechanisms implicated are however poorly understood, although recent progresses have been made to identify key components in the CKD process. We will present results on the effect of uremic toxins on erythropoiesis, having an impact on cell metabolism during this process. Taken together, our findings could be of interest to both researchers and clinicians working in the field since they might shed new light on the molecular mechanisms involved in the CKD process. MicroRNAs implicated in Chronic Kidney Disease Pr. Laurent Metzinger, UR-UPJV 4666 HEMATIM, CURS, Université de Picardie Jules Verne, CHU Amiens Sud, Avenue René Laënnec, Salouel, F-80054, Amiens, France. Tel: (+33) 22 82 53 56, Email: laurent.metzinger@u-picardie.fr

Three-dimensional nanorod array for label-free surface-enhanced Raman spectroscopy analysis of microRNA pneumoconiosis biomarkers

Novel approaches are required to overcome the challenges associated with conventional microRNA (miRNA) detection methods and realize the early diagnosis of diseases. This work describes a novel label-free surface-enhanced Raman spectroscopy (SERS) method for the detection of the miRNA biomarkers for pneumoconiosis on a three-dimensional Au-coated ZnO nanorod array (Au-ZnO NRA). The Au-ZnO NRA substrate, which was fabricated via a modified seeding method combined with ion sputtering, provided a high enhancement factor and good spatial uniformity of the signal. With the Au-ZnO NRA, the SERS spectra of miRNAs were obtained in 30 s without labeling at room temperature. Density functional theory calculations were performed to understand the structural fingerprints of the miRNAs. Principal component analysis was carried out to identify the pneumoconiosis biomarkers based on their fingerprint SERS signals. Dual-logarithm linear relationships between the SERS intensity and the miRNA concentration were proposed for quantitative analysis. The label-free SERS method has limits of detection on the femtomolar level, which is much lower than the concentrations of the miRNA biomarkers for pneumoconiosis in lung fibroblasts.

Circulating microRNAs 34a, 122, and 192 are linked to obesity-associated inflammation and metabolic disease in pediatric patients

BackgroundObesity-associated chronic low-grade inflammation leads to dysregulation of central lipid and glucose metabolism pathways leading to metabolic disorders. MicroRNAs (miRNAs) are known to control regulators of metabolic homeostasis. We aimed to assess the relationship of circulating miRNAs with inflammatory modulators and metabolic disorders in pediatric obesity.MethodsFrom a pediatric cohort with severe obesity (n = 109), clinically thoroughly characterized including diverse routine blood parameters, oral glucose tolerance test, and liver MRI, a panel of 16 circulating miRNAs was quantified using qRT-PCR. Additionally, markers of inflammation TNFα, IL1 receptor antagonist, procalcitonin, CRP, and IL-6 were measured.ResultsMarkers of obesity-associated inflammation, TNFα, IL-1Ra, and procalcitonin, all significantly correlated with concentrations of miRNAs 122 and 192. Concentrations of these miRNAs negatively correlated with serum adiponectin and were among those strongly linked to parameters of dyslipidemia and liver function. Moreover, miRNA122 concentrations correlated with HOMA-IR. Several miRNA levels including miRNAs 34a, 93, 122, and 192 were statistically significantly differing between individuals with prediabetes, impaired glucose tolerance, metabolic syndrome, or nonalcoholic fatty liver disease compared to the respective controls. Additionally, miRNA 192 was significantly elevated in metabolically unhealthy obesity.ConclusionsA miRNA pattern associated with obesity-associated inflammation and comorbidities may be used to distinguish metabolically healthy from unhealthy pediatric patients with obesity. Moreover, these changes in epigenetic regulation could potentially be involved in the etiology of obesity-linked metabolic disease in children and adolescents.

Bioengineered miRNA Agents are More Efficacious and Selective than Chemo‐Engineered Mimics in the Regulation of Target Gene Expression

Recent investigations have demonstrated the utility of small interfering RNAs (siRNA) and microRNAs (miRNA) as therapeutics for the treatment of many diseases including cancers, viral infections, cardiovascular diseases, and many other disorders. However, current RNAi research and development is limited to the use of chemically-synthesized RNA agents that are mounted with various artificial modifications. In this study, we aimed to optimize the tRNA/pre-miRNA-based technology to achieve in vivo fermentation production of fully-humanized bioengineered RNAi agents (hBERA). We first achieved high-level heterogenous overexpression (accounting for >50% of total bacterial RNA) of 20 target hBERAs at 100% success rate, yielding 18-53 mg high-purity (≥98.5%) and low endotoxin (<1.5 EU/µg RNA) hBERAs per liter bacterial culture. Further studies demonstrated that target miRNAs (e.g., miR-124-3p) are specifically released from hBERAs in human NSCLC cells to modulate protein levels of targeted genes (e.g., VAMP3 and IQGAP1) and control cell viability. Interestingly, hBERA/miRNAs were more effective and selective than the same concentrations of synthetic miRNA mimics in the control of target gene expression and human NSCLC cell viability. In addition, systemic administration of lipid nanoparticle formulated hBERA had no significant effects on cytokine release in both human PBMCs and immunocompetent mice, as well as hepatic and renal functions. This robust RNA bioengineering platform offers a novel class of fully-humanized, true biologic RNAi agents that are more efficacious than synthetic RNA mimics for research and may be developed as new therapeutics.

Chemical binding of pyrrolidinyl peptide nucleic acid (acpcPNA-T9) probe with AuNPs toward label-free monitoring of miRNA-21: A novel biosensing platform for biomedical analysis and POC diagnostics.

miRNAs are attractive factors in cancer research studies due to their important roles for regulating of gene expression. Because of miRNA-21 expression surplus in many types of cancers, so accurate identification is important. Increasing efforts have caused different methods to improve the sensitivity and specificity of detection. Present study is an attempt to report a new electrochemical label-free PNA-based bioassay for detection of miRNA-21. In this study, gold electrode was modified by gold nanoparticles to improve a functional PNA-based biosensor. The EDS and field emission scanning electron microscope (FE-SEM) were used to detect fabrication of biosensor. The electrochemical behavior of sensor was evaluated after inserting of acpcPNA probes and miRNA-21 on the stucture of electrode and analyzed essential parameters such as various concentration of target miRNA, hybridization time, reproducibility, stability, and applicability. The results of study demonstrated that engineered biosensor was successfully fabricated. The findings showed the highest amount of current in 5 minutes hybridization time, with suitable reproducibility and stability. This innovative miRNA-based biosensor presents a sensitive and specific method in fast and may be lab-on chip assay in future.

Multichannel Paper Chip-Based Gas Pressure Bioassay for Simultaneous Detection of Multiple MicroRNAs.

Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.

Detection of Cervical Lesions and Cancer in Air-Dried Cytologic Smears by Combined Analysis of mRNA and miRNA Expression Levels

Cervical cancer screening is based on cytologic analysis and high-risk human papillomavirus (HR-HPV) testing, each having their drawbacks. Implementation of new biomarker-based methods may improve screening accuracy. Here, the levels of 25 microRNAs (miRNAs, miRs) and 12 mRNAs involved in cervical carcinogenesis in 327 air-dried Papanicolaou-stained cervical smears from patients with cervical precancerous lesions, cancer, or without the disease were estimated by real-time PCR. Using logistic regression analysis, small-scale miRNA-based, mRNA-based, and combined molecular classifiers were built based on paired ratios of miRNA or mRNA concentrations; their ability to detect high-grade cervical lesions and cancer was then compared. Combined mRNA-miRNA classifiers manifested a better combination of sensitivity and specificity than miRNA- and mRNA-based classifiers. The best classifier, combining miR-375, miR-20, miR-96, CDKN2A, TSP4, and ECM1, predicted high-grade lesions with diagnostic sensitivity of 89.0%, specificity of 84.2%, and a receiver-operating characteristic area under the curve of 0.913. Additionally, in a subsample of the same specimens, the levels of MIR124-2 and MAL promoter methylation, HR-HPV genotypes, and viral loads were analyzed. The relative high-grade lesion risk estimated by the classifier correlated with the frequency of MAL and MIR124-2 methylation but not with the HR-HPV genotype or viral load. The results support the feasibility of cellular biomarker-based methods for cervical screening and patient management.

Construction of microRNA-21 terahertz metamaterial sensing method based on duplex-specific nuclease triggered rolling circle amplification

Objective: To construct of a terahertz metamaterial sensing method for detection of microRNA-21 (miRNA-21) based on duplex-specific nuclease triggered rolling circle amplification. Methods: First, a THz metamaterial sensing method was constructed, and verified by polyacrylamide gel electrophoresis and zeta potential; after optimizing the detection conditions of the sensor, different concentrations of miRNA-21 and other different miRNAs were detected. And this biosensor was compared with other miRNA detection methods; finally, the recovery rate of the biosensor was evaluated. Results: Under the optimal experimental conditions, through the dual signal amplification strategy of duplex-specific nuclease (DSN) cycle recognition and rolling circle amplification (RCA), the THz metamaterial sensor has a response range of 10 fmol/L to 10 nmol/L to the target miRNA-21, with a detection limit of 8.49 fmol/L. And the biosensor has good specificity with the ability to recognize the target miRNA-21 from a variety of microRNAs. And the experiment with recovery rate from 94.33% to 115.33% has been further verified in commercial human serum samples. Conclusion: The terahertz biosensor can achieve highly sensitive and specific detection of the target miRNA-21, which proves the potential for label-free diagnosis and early warning of miRNA-related diseases.

Predictive Value of Circulating miRNAs in Lymph Node Metastasis for Colon Cancer.

(1) Background: Lymph node (LN) status is an indubitable prognostic factor for survival among colon cancer patients. MicroRNAs (miRNAs) have been implicated in the development and progression of many cancers and are potential biomarkers for cancer diagnosis and prognosis. Therefore, we validated candidate biomarkers using circulating miRNAs by analyzing the plasma miRNA concentrations from patients with colon cancer to predict LN metastasis. (2) Methods: This study included 79 blood samples from patients diagnosed with colon cancer. The NanoString assay was used for screening, and TaqMan miRNA assays for quantitative real-time polymerase chain reaction (RT-PCR) test was used for validation. In a discovery set, we compared the expression of 800 circulating miRNAs in 24 samples (stage 0/I/IIA versus IIIB/IIIC). For validation, a total 79 samples were tested using quantitative RT-PCR. (3) Results: In the discovery set, 10 candidate circulating miRNAs were detected (4 up-regulated miRNAs: miR-323a-3p, miR-382-5p, miR-29a-3p, and miR-376a-3p; 6 down-regulated miRNAs: miR-26a-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-374a-5p, and let-7b-5p). In the validation set, higher expression of three circulating miRNAs (miR-323a-3p, miR-382-5p, and miR-376a-3p) was significantly associated with LN metastasis (p = 0.0063, 0.0107, and 0.0022). (4) Conclusions: High expression of circulating miR-323a-3p, miR-382-5p, and miR-376a-3p was significantly associated with LN metastasis in colon cancer patients. These miRNAs could be circulating biomarker candidates that predict the presence of LN metastasis.

Thermal Ablation and Transarterial Chemoembolization are Characterized by Changing Dynamics of Circulating MicroRNAs

PurposeTo determine whether the levels of circulating microRNAs (miRNAs) are altered in patients undergoing thermal ablation and chemoembolization and whether these changes are predictive of a clinical outcome. Material and MethodsThis prospective study consisted of 43 patients diagnosed with hepatocellular carcinoma (n = 15) and intrahepatic colorectal cancer metastases (n = 28) treated with thermal ablation (n = 23; radiofrequency [n = 6] or microwave [n = 19]), chemoembolization using drug-eluting embolics (n = 18), or both (n = 2). Four blood samples (immediately before the intervention and 60–90 minutes, 24 hours, and 7 days after the intervention) were taken to measure the plasma concentrations of miRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122), epithelial–mesenchymal transition (miR-200a), and apoptosis (miR-34a) using miRNA-specific TaqMan assays and quantitative real-time polymerase chain reaction. Tumor burden and treatment response at 3 months were evaluated using the modified response evaluation criteria in solid tumors. The miRNA results were compared with clinical outcomes (Mann-Whitney U test, Wilcoxon matched-pair test). ResultsDynamic changes in the circulating miRNA levels were observed following both the interventions. For thermal ablation, significant increases in miR-21, miR-210, miR-122, miR-200a, and miR-34a concentrations peaked 60–90 minutes after the intervention (P < .01). However, for transarterial chemoembolization, maximum increases in the miRNA concentrations were observed at 24 hours after the intervention for miR-21, miR-210, miR-122, miR-200a, and miR-34a (P < .05). The increased concentrations of the circulating miRNAs were followed by a subsequent decline to baseline by 7 days. For the thermal ablation (but not chemoembolization) patients, elevations in the miR-210 and miR-200a levels were associated with early progressive disease at 3 months (P = .040 and P = .012, respectively). ConclusionsIncreased but dynamic levels of circulating miRNAs are present following interventional oncologic procedures and may prove useful as biomarkers for the monitoring of clinical outcomes.

Algorithm-Assisted Detection and Imaging of microRNAs in Living Cancer Cells via the Disassembly of Plasmonic Core-Satellite Probes Coupled with Strand Displacement Amplification.

Acute detection and high-resolution imaging of microRNAs (miRNAs) in living cancer cells have attracted great attention in clinical diagnosis and therapy. However, current methods suffer from low detection sensitivity or heavy dependence on expensive and sophisticated spectrometers. Herein, a novel algorithm-assisted system of detecting and imaging miRNAs in living cancer cells was developed via the disassembly of plasmonic core-satellite probes coupled with strand displacement amplification (SDA). The target miRNAs in the system could trigger the disassembly of plasmonic core-satellite probes, leading to the color change in the scattering light of the probes, which could be captured by dark-field microscopy (DFM). The concentration of the target miRNAs was obtained by analyzing the dark-field image based on the proposed algorithm with a detection limit of 2 pM for miRNA-21. Thus, the performance in terms of simplicity and sensitivity of the system compared with one of the conventional spectrophotometers was well presented, which could inspire more clinical applications of inexpensive, intelligent, and rapid screening of cancer cells. The application software based on the proposed algorithm running on the Android platform was also developed, demonstrating the potential of remote diagnosis.

Photoinduced-electron transfer coupled with DNA cross-chain displacement multiple amplification for fluorescence biosensing of MicroRNA

In this work, a new fluorescence biosensor platform based on distance-dependent photoinduced-electron transfer (PET) coupled with target cross-chain displacement cyclic amplification strategy was developed to detect MicroRNA. The DNA cross structure was cleverly designed to protect restriction site, then multiple amplification reactions of target cycle and chain replacement based on DNA cross-configuration were carried out in the presence of primer, polymerase and cutting enzyme, thus a large number of single-stranded (ss) DNA products (S1 and S2) can be exported by inputting a small amount of target miRNA. The fluorescent AgNCs/DNA probe was synthesized based on high affinity of Ag to cytosine (C) rich in ssDNA acting as electron donor, and guanine (G) rich ssDNA can form G-quadruplex complex acting as electron receptor to induce PET process. S1 and S2 hybridized with flexible single-stranded DNA COM 1 and Com 2, forming rigid double-stranded DNA to inhibit fluorescence quenching PET process, so the corresponding fluorescence was recovered. Thus the miRNA-induced amplified products can specifically result in fluorescence changes by PET, and the changes increase with increasing miRNA concentration. Therefore, the proposed fluorescent biosensor can be applied to quantitative determination of miRNA-182-5p, which has great potential in early clinical diagnosis of miRNAs related diseases.

Plasma miRNAs improve the prediction of coronary atherosclerosis in patients with rheumatoid arthritis.

MicroRNAs (miRNAs) regulate gene expression and are disease biomarkers. Rheumatoid arthritis (RA) patients have accelerated atherosclerosis leading to excess cardiovascular morbidity and mortality, but traditional risk factors for cardiovascular risk stratification are inadequate. In the general population, miRNAs improve cardiovascular risk estimation beyond traditional risk factors. Our objective was to develop a miRNA panel that predicts coronary atherosclerosis in RA patients. Plasma small RNA next-generation sequencing (NGS) was performed on 161 RA patients whose Agatston scores for coronary artery calcium were previously measured. Random forest analysis of plasma NGS miRNA expression was used to determine which miRNAs best differentiated between those patients with and without coronary artery calcium. Top predictive miRNAs were assayed by quantitative PCR (qPCR). Elastic net regression was used to develop the most parsimonious models with qPCR-measured miRNA concentrations and clinical variables (age, sex, ACC/AHA 10-year risk score, DAS28 score, and diabetes) separately to predict the presence of coronary artery calcium and high coronary artery calcium. C-statistics were used to assess performance model performance. The top miRNAs which differentiated those with and without coronary atherosclerosis based on random forest analysis included let-7c-5p, miR-30e-5p, miR-30c-5p, miR-4446-3p, miR-126-5p, miR-3168, miR-425-5p, miR-126-3p, miR-30a-5p, and miR-125a-5p. For coronary artery calcium prediction, addition of all miRNAs except miR-126-3p to clinical factors improved the c-statistic modestly from 0.86 to 0.87. For high coronary artery calcium prediction, addition of all miRNAs except miR-30c-5p to clinical factors improved the c-statistic from 0.75 to 0.80. A plasma miRNA panel improved the prediction of high coronary artery calcium beyond traditional risk factors and RA disease activity. Further evaluation of the miRNA panel for prediction of coronary events in RA is necessary. Key Point • A plasma microRNA panel including let-7c-5p, miR-30a-5p, miR-30e-5p, miR-125a-5p, miR-126-3p, miR-126-5p, miR-425-5p, miR-3168, and miR-4446-3p improved the prediction of high coronary artery calcium beyond clinical factors in patients with rheumatoid arthritis.

Behind the Scene: Surprises and Snags of Pseudogenes.

The junk DNA "pseudogenes," known as genomic fossils, are characterized by their ubiquitousness and abundance within the genomic structure. These genomics sets are recognized by the potential activity of meta-regulating the parent genes; these are transcribed into interfering RNA, consequently acting on miRNA concentration, thereby shedding light on the crosstalk of the pseudogenes' miRNA, siRNA, lncRNA/tumor therapy co-relationship. Moreover, an upcoming visualization regarding pseudogenes is under investigation, which describes the potentiality of pseudogenes as a fundamental component of cancerous evolutionary processing tools. Accordingly, here is a systematic review covering pseudobirth, pseudosignatures, and functional properties of pseudogenes, concluding that these pseudogenes are hypothetically predictive tumor therapies.

Magnetic nanoparticle-based amplification of microRNA detection in body fluids for early disease diagnosis.

Circulating biomarkers such as microRNAs (miRNAs), short noncoding RNA strands, represent prognostic and diagnostic indicators for a variety of physiological disorders making their detection and quantification an attractive approach for minimally invasive early disease diagnosis. However, highly sensitive and selective detection methods are required given the generally low abundance of miRNAs in body fluids together with the presence of large amounts of other potentially interfering biomolecules. Although a variety of miRNA isolation and detection methods have been established in clinics, they usually require trained personnel and often constitute labor-, time- and cost-intensive approaches. During the last years, nanoparticle-based biosensors have received increasing attention due to their superior detection efficiency even in very low concentration regimes. This is based on their unique physicochemical properties in combination with their high surface area that allows for the immobilization of multiple recognition sites resulting in fast and effective recognition of analytes. Among various materials, magnetic nanoparticles have been identified as useful tools for the separation, concentration, and detection of miRNAs. Here, we review state-of-the-art technology with regard to magnetic particle-based miRNA detection from body fluids, critically discussing challenges and future perspective of such biosensors while comparing their handling, sensitivity as well as selectivity against the established miRNA isolation and detection methods.

MiRNA Expression in Patients with Gaucher Disease Treated with Enzyme Replacement Therapy

Aims: The aim of the work was to establish potential biomarkers or drug targets by analysing changes in miRNA concentration among patients with Gaucher disease (GD) compared to in healthy subjects. Methods: This study was an observational, cross-sectional analysis of 30 adult participants: 10 controls and 20 adults with GD type 1. Patients with GD type 1 were treated with enzyme replacement therapy (ERT) for at least two years. The control group was composed of healthy volunteers, unrelated to the patients, adjusted with age, sex and body mass index (BMI). The miRNA alteration between these groups was examined. After obtaining preliminary results on a group of six GD patients by the high-output method (TaqMan low-density array (TLDA)), potential miRNAs were selected for confirming the results by using the qRT-PCR method. With Diane Tools, we analysed miRNAs of which differential expression is most significant and their potential role in GD pathophysiology. We also determined the essential pathways these miRNAs are involved in. Results: 266 dysregulated miRNAs were found among 753 tested. Seventy-eight miRNAs were downregulated, and 188 were upregulated. Thirty miRNAs were significantly altered; all of them were upregulated. The analysis of pathways regulated by the selected miRNAs showed an effect on bone development, inflammation or regulation of axonal transmission in association with Parkinson’s disease. Conclusions: We revealed few miRNAs, like miR-26-5p, which are highly altered and fit the GD pathophysiological model, might be considered as novel biomarkers of disease progression but need further evaluation.

Profiling microRNAs in uncomplicated pregnancies: Serum vs. plasma.

Blood-derived microRNAs (miRNAs/miRs) are ideal clinical biomarkers, as they can be relatively non-invasively extracted and are stable across a range of storage conditions. However, the concentration and profile of miRNAs differ between specific patient groups and starting media, which must be a key consideration before embarking upon uses for clinical applications. The optimum blood-derived starting media for biomarker discovery involving pregnant women with an uncomplicated pregnancy has not been determined. Paired serum and plasma samples were collected from 10 pregnant women with uncomplicated low-risk pregnancies at three time points: i) During the second trimester of pregnancy; ii) during the third trimester; and iii) 6 weeks post-partum. Sample miRNA content was assessed using an Agilent Bioanalyzer Small RNA chip and reverse transcription-quantitative (RT-q)PCR using four constitutively expressed miRNAs: hsa-miR-222-3p, hsa-miR-23a, hsa-miR-30e-5p and hsa-miR-451a. Quality control spike-ins measured RNA extraction (UniSp2) and cDNA extraction (cel-miR-39-3p) efficiency. MiRNA concentration and percentage were significantly higher in the serum vs. plasma samples based on data obtained from the Bioanalyzer; however, RT-qPCR failed to replicate these differences in the majority of comparisons using the ΔCq values of the four constitutively expressed miRNAs. Using the standard deviations of the ΔCq values, the consistency of serum and plasma in terms of miRNA expression levels were equivalent. Thus, clinicians and researchers should take into consideration that different miRNA quantification methods can yield contrasting results with regards to the starting media utilized. Based on the equivalent performance of serum and plasma assessed using RT-qPCR, which is less likely to be influenced by the coagulation process or degraded long RNAs, both starting media assessed in the present study are equally suitable for ongoing biomarker discovery studies involving healthy pregnant women at any gestational time point or immediately postpartum.