miRNA-based Diagnostics Research Articles

Understanding microRNAs in the context of bacterial versus viral infections.

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have emerged as biomarkers for differentiating infection types due to their distinct expression profiles in response to pathogens. This study explores miRNA profiling using microarray technology to identify miRNA signatures that differentiate viral from bacterial infections in plasma samples. Plasma samples were collected from patients diagnosed with either bacterial (e.g., pneumonia) or viral (e.g., human papillomavirus) infections; control samples were used to evaluate altered miRNA pattern, followed by Ingenuity Pathway Analysis (IPA) analysis. Microarray analysis revealed distinct miRNA expression patterns for bacterial and viral infections. In bacterial infections, 11 miRNAs were significantly downregulated compared to controls. Similarly, 12 miRNAs were downregulated in viral infections. Pathway analysis indicated that the altered miRNAs in bacterial infections were linked to immune and inflammatory pathways. In contrast, viral infections were associated with miRNAs involved in cellular stress and replication processes. Plasma miRNA profiling offers a promising diagnostic tool to differentiate bacterial from viral infections, providing specific miRNA signatures underlying immune responses. These findings represent a foundation for developing miRNA-based diagnostics, improving the precision of infection diagnosis, and paving the way for targeted therapeutic interventions.

Localized hybridization chain reaction amplifier utilizing three-dimensional DNA nanostructures for efficient and reliable microRNA imaging in living cells

MicroRNAs (miRNAs) are pivotal in regulating biological processes such as cell proliferation and disease progression. Traditional miRNA detection methods like qRT-PCR and Northern blotting do not allow for monitoring dynamic changes in living cells and typically require invasive sample collection. This research presents a robust, non-enzymatic technique known as the Localized Hybridization Chain Reaction Amplifier (LHCRA), designed for real-time, in vivo miRNA imaging. This method addresses these limitations by providing rapid and sensitive detection of miRNAs, crucial for progressing clinical diagnostics and research. The LHCRA utilizes hairpin chain reaction probes embedded within self-assembled DNA micelles (DM), significantly amplifying miRNA signals. LHCRA demonstrated exceptional performance, with a detection limit of 100 pM and a response time of 10 min. Importantly, it effectively differentiated between cancerous and normal cells using clinical peripheral blood samples, showcasing high specificity and stability under physiological conditions. Additionally, LHCRA maintained consistent performance in complex biological media, including 10 % serum and 2 U/mL DNase I, confirming its broad applicability in various diagnostic scenarios. This performance highlights LHCRA's potential as a transformative tool in miRNA-based diagnostics. The introduction of LHCRA marks a significant advancement in miRNA detection technology. By enabling accurate, non-invasive, and real-time monitoring of miRNA dynamics within living cells, this method offers substantial potential to enhance diagnostic accuracy and deepen our understanding of disease mechanisms, particularly in cancer. Thus, it could greatly improve therapeutic strategies and patient outcomes in clinical environment.

Exploring the utility of circulating miRNAs as diagnostic biomarkers of fasciolosis

Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.

MicroRNAs: Small but Key Players in Viral Infections and Immune Responses to Viral Pathogens.

Since the discovery of microRNAs (miRNAs) in C. elegans in 1993, the field of miRNA research has grown steeply. These single-stranded non-coding RNA molecules canonically work at the post-transcriptional phase to regulate protein expression. miRNAs are known to regulate viral infection and the ensuing host immune response. Evolving research suggests miRNAs are assets in the discovery and investigation of therapeutics and diagnostics. In this review, we succinctly summarize the latest findings in (i) mechanisms underpinning miRNA regulation of viral infection, (ii) miRNA regulation of host immune response to viral pathogens, (iii) miRNA-based diagnostics and therapeutics targeting viral pathogens and challenges, and (iv) miRNA patents and the market landscape. Our findings show the differential expression of miRNA may serve as a prognostic biomarker for viral infections in regard to predicting the severity or adverse health effects associated with viral diseases. While there is huge market potential for miRNA technology, the novel approach of using miRNA mimics to enhance antiviral activity or antagonists to inhibit pro-viral miRNAs has been an ongoing research endeavor. Significant hurdles remain in terms of miRNA delivery, stability, efficacy, safety/tolerability, and specificity. Addressing these challenges may pave a path for harnessing the full potential of miRNAs in modern medicine.

SAT047 Regulation Of The Insulin Receptor And The Insulin-like Growth Factor 1 Receptor By miR-322-5p, A miR-16 Family Member, In Hypothalamic Neuronal Models

Abstract Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

Self-Assembled Monolayers Derived from Positively Charged Adsorbates on Plasmonic Substrates for MicroRNA Delivery: A Review

MicroRNA (miRNA) has emerged as a promising alternative therapeutic treatment for cancer, but its delivery has been hindered by low cellular uptake and degradation during circulation. In this review, we discuss the various methods of delivering miRNA, including viral and non-viral delivery systems such as liposomes and nanoparticles. We also examine the use of nanoparticles for miRNA-based diagnostics. We focus specifically on non-viral delivery systems utilizing coinage metals in the form of nanoparticles and the use of self-assembled monolayers (SAMs) as a method of surface modification. We review the use of SAMs for the conjugation and delivery of small noncoding ribonucleic acid (ncRNA), particularly SAMs derived from positively charged adsorbates to generate charged surfaces that can interact electrostatically with negatively charged miRNA. We also discuss the effects of the cellular uptake of gold and other plasmonic nanoparticles, as well as the challenges associated with the degradation of oligonucleotides. Our review highlights the potential of SAM-based systems as versatile and robust tools for delivering miRNA and other RNAs in vitro and in vivo and the need for further research to address the challenges associated with miRNA delivery and diagnostics.

Selecting optimum miRNA panel for miRNA signature-based companion diagnostic model to predict the response of R-CHOP treatment in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. Although the first-line treatment, R-CHOP treatment, shows efficacy in approximately 80% of patients with DLBCL, some patients have refractory disease or relapse after the initial response to therapy, resulting in a significantly poorer prognosis. In this study, we developed a microRNA (miRNA) signature-based companion diagnostic model to predict the response of patients with DLBCL to R-CHOP treatment by integrating two clinical study datasets. To select the optimum miRNA combination as a panel, we examined three feature selection methods (p-value-based ranking, stepwise method, and Boruta), together with 11 types of classifiers systematically. Boruta selection enabled a higher area under the curve (AUC) with a lower number of miRNAs compared with other feature selection methods, leading to an AUC of 0.751 via the random forest classifier using 36 miRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that Boruta avoided multiple selection of miRNAs with similar functions, thereby preventing the decrease in diagnostic ability via collinearity. The AUC value first increased with an increasing number of miRNAs and then became almost constant at approximately 30 miRNAs, suggesting the existence of the optimum number of miRNAs as a panel for future clinical translation of multiple miRNA-based diagnostics.

Analysis of Overexpressed miRNA in Circulation and Cancer Tissue to Develop a Potential microRNA Panel for the Diagnosis of Colorectal Cancer.

Colorectal cancer (CRC) represents the world's fourth deadly cancer, but its early diagnosis can be curative with considerable success rates. This study was aimed to identify CRC-specific microRNAs (miRNAs) in tissue and serum samples to develop a miRNA-based diagnostics panel for the minimal invasive detection of CRC in early conditions. By integrating four microarrays in tissue and serum samples of CRC from the Gene Expression Omnibus (GEO) database, we screened out the highly expressed miRNAs in each dataset using the limma R package. Two important upregulated miRNAs, namely hsa-miR-1246 and hsamiR- 1825, were overlapped in both tissue and serum samples of CRC and were investigated to target identification, followed by functional annotation and protein-protein interaction (PPI) study for the target genes through DAVID and STRING, respectively. Finally, hub target genes were retrieved by Cytoscape analysis. It was shown that target genes of hsa-miR-1246 and hsa-miR-1825 were involved with core KEGG pathways (such as cAMP, PI3K-Akt, and calcium signaling pathway). In addition, biological processes (such as cell adhesion and cell proliferation), cellular components (such as plasma membrane and cytosol), molecular functions (such as protein binding and metal ion binding) were mostly associated with the target genes. Their top 5 target genes were retrieved, and their biological function towards tumor progression was shown using Cancer Hallmarks Analytics Tool. This study suggested that hsa-miR-1246 and hsa-miR-1825, as overlapped upregulated tissue and circulating miRNAs, might have a vital role in the development of CRC, and their five hub target genes were identified.

Cervical cancer development, chemoresistance, and therapy: a snapshot of involvement of microRNA.

Cervical cancer (CC) is one of the leading causes of death in women due to cancer and a major concern in the developing world. Persistent human papilloma virus (HPV) infection is the major causative agent for CC. Besides HPV infection, genetic and epigenetic factors including microRNA (miRNA) also contribute to the malignant transformation. Earlier studies have revealed that miRNAs participate in cell proliferation, invasion and metastasis, angiogenesis, and chemoresistance processes by binding and inversely regulating the target oncogenes or tumor suppressor genes. Based on functions and mechanistic insights, miRNAs have been identified as cellular modulators that have an enormous role in diagnosis, prognosis, and cancer therapy. Signatures of miRNA could be used as diagnostic markers which are necessary for early diagnosis and management of CC. The therapeutic potential of miRNAs has been shown in CC; however, more comprehensive clinical trials are required for the clinical translation of miRNA-based diagnostics and therapeutics. Understanding the molecular mechanism of miRNAs and their target genes has been useful to develop miRNA-based therapeutic strategies for CC and overcome chemoresistance. In this review, we summarize the role of miRNAs in the development, progression, and metastasis of CC as well as chemoresistance. Further, we discuss the diagnostic and therapeutic potential of miRNAs to overcome chemoresistance and treatment of CC.

MicroRNAs in Prion Diseases-From Molecular Mechanisms to Insights in Translational Medicine.

MicroRNAs (miRNAs) are small non-coding RNA molecules able to post-transcriptionally regulate gene expression via base-pairing with partially complementary sequences of target transcripts. Prion diseases comprise a singular group of neurodegenerative conditions caused by endogenous, misfolded pathogenic (prion) proteins, associated with molecular aggregates. In humans, classical prion diseases include Creutzfeldt–Jakob disease, fatal familial insomnia, Gerstmann–Sträussler–Scheinker syndrome, and kuru. The aim of this review is to present the connections between miRNAs and prions, exploring how the interaction of both molecular actors may help understand the susceptibility, onset, progression, and pathological findings typical of such disorders, as well as the interface with some prion-like disorders, such as Alzheimer’s. Additionally, due to the inter-regulation of prions and miRNAs in health and disease, potential biomarkers for non-invasive miRNA-based diagnostics, as well as possible miRNA-based therapies to restore the levels of deregulated miRNAs on prion diseases, are also discussed. Since a cure or effective treatment for prion disorders still pose challenges, miRNA-based therapies emerge as an interesting alternative strategy to tackle such defying medical conditions.

Isolation of Extracellular Vesicles from Biological Fluids via the Aggregation-Precipitation Approach for Downstream miRNAs Detection.

Extracellular vesicles (EVs) have high potential as sources of biomarkers for non-invasive diagnostics. Thus, a simple and productive method of EV isolation is demanded for certain scientific and medical applications of EVs. Here we aim to develop a simple and effective method of EV isolation from different biofluids, suitable for both scientific, and clinical analyses of miRNAs transported by EVs. The proposed aggregation–precipitation method is based on the aggregation of EVs using dextran blue and the subsequent precipitation of EVs using 1.5% polyethylene glycol solutions. The developed method allows the effective isolation of EVs from plasma and urine. As shown using TEM, dynamic light scattering, and miRNA analyses, this method is not inferior to ultracentrifugation-based EV isolation in terms of its efficacy, lack of inhibitors for polymerase reactions and applicable for both healthy donors and cancer patients. This method is fast, simple, does not need complicated equipment, can be adapted for different biofluids, and has a low cost. The aggregation–precipitation method of EV isolation accessible and suitable for both research and clinical laboratories. This method has the potential to increase the diagnostic and prognostic utilization of EVs and miRNA-based diagnostics of urogenital pathologies.

Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification.

MicroRNAs (miRNAs) play crucial regulatory roles as post-transcriptional regulators for gene expression and serve as promising biomarkers for diagnosis and prognosis of diseases. Herein, a dual-signal amplification method has been developed for sensitive and selective detection of miRNA based on rolling circle amplification (RCA) and enzymatic repairing amplification (ERA) with low nonspecific background. This strategy designs a padlock probe that can be cyclized in the presence of target miRNA to initiate the RCA reaction, after which the TaqMan probes that are complementary to the RCA products can be cyclically cleaved to produce obvious fluorescence signals with the help of endonuclease IV (Endo IV). Attributed to the dual-signal amplification procedure and the high fidelity of Endo IV, the RCA–ERA method allows quantitative detection of miR-21 in a dynamic range from 2 pM to 5 nM with a low background signal. Moreover, it has the ability to discriminate single-base difference between miRNAs and shows good performance for miRNA detection in complex biological samples. The results demonstrate that the RCA–ERA assay holds a great promise for miRNA-based diagnostics.

MiRACLe: an individual-specific approach to improve microRNA-target prediction based on a random contact model.

Deciphering microRNA (miRNA) targets is important for understanding the function of miRNAs as well as miRNA-based diagnostics and therapeutics. Given the highly cell-specific nature of miRNA regulation, recent computational approaches typically exploit expression data to identify the most physiologically relevant target messenger RNAs (mRNAs). Although effective, those methods usually require a large sample size to infer miRNA-mRNA interactions, thus limiting their applications in personalized medicine. In this study, we developed a novel miRNA target prediction algorithm called miRACLe (miRNA Analysis by a Contact modeL). It integrates sequence characteristics and RNA expression profiles into a random contact model, and determines the target preferences by relative probability of effective contacts in an individual-specific manner. Evaluation by a variety of measures shows that fitting TargetScan, a frequently used prediction tool, into the framework of miRACLe can improve its predictive power with a significant margin and consistently outperform other state-of-the-art methods in prediction accuracy, regulatory potential and biological relevance. Notably, the superiority of miRACLe is robust to various biological contexts, types of expression data and validation datasets, and the computation process is fast and efficient. Additionally, we show that the model can be readily applied to other sequence-based algorithms to improve their predictive power, such as DIANA-microT-CDS, miRanda-mirSVR and MirTarget4. MiRACLe is publicly available at https://github.com/PANWANG2014/miRACLe.

MicroRNAs As Master Regulators of Atherosclerosis: From Pathogenesis to Novel Therapeutic Options.

Significance: Cardiovascular disease (CVD) remains the major cause of morbidity and mortality worldwide. Accumulating evidence indicates that atherosclerosis and its sequelae, coronary artery disease, contribute to the majority of cardiovascular deaths. Atherosclerosis is a chronic inflammatory disease of the arteries in which atherosclerotic plaques form within the vessel wall. Epidemiological studies have identified various risk factors for atherosclerosis, such as diabetes, hyperlipidemia, smoking, genetic predisposition, and sedentary lifestyle. Recent Advances: Through the advancement of genetic manipulation techniques and their use in cardiovascular biology, it was shown that small RNAs, especially microRNAs (miRNAs), are dynamic regulators of disease pathogenesis. They are considered to be central during the regulation of gene expression through numerous mechanisms and provide a means to develop biomarkers and therapeutic tools for the diagnosis and therapy of atherosclerosis. Circulating miRNAs encapsulated within membrane-surrounded vesicles, which originate from diverse subcellular compartments, are now emerging as novel regulators of intercellular communication. The miRNAs, in both freely circulating and vesicle-bound forms, represent a valuable tool for diagnosing and monitoring CVD, recently termed as "liquid biopsy." Critical Issues: However, despite the recent advancements in miRNA-based diagnostics and therapeutics, understanding how miRNAs can regulate atherosclerosis is still crucial to achieving an effective intervention and reducing the disease burden. Future Directions: We provide a landscape of the current developmental progression of RNA therapeutics as a holistic approach for treating CVD in different animal models and clinical trials. Future interrogations are warranted for the development of miRNA-based therapeutics to overcome challenges for the treatment of the disease.

Abstract LB-388: New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase

Abstract miRNAs have an immense potential to serve as diagnostic, prognostic and prediction biomarkers in the whole field of oncology. Their utilization in liquid biopsy as a non-invasive diagnostic tool is very promising as well. However, to our knowledge, no miRNA-based diagnostics is currently used in clinical laboratories. We believe the reason is simple - the lack of easy, fast, sensitive and reproducible measurement technology, which would have passed the strict clinical validation criteria. On this count we have optimized a very promising miRNA detection technique, utilizing the enzyme Chlorella virus DNA ligase (SplintR® ligase, New England Biolabs), for clinical use. This two-step method involves ligation of two adjacent DNA oligonucleotides hybridized to a miRNA target, followed by TaqMan probe real-time quantitative PCR (qPCR). We managed to reduce the total procedure time from previous 5 hours to 2 hours and 15 minutes maintaining the high sensitivity (1 amol/µl in original clinical sample) and large dynamic range (7 logs). Moreover, we enabled the use of two PCR detection chemistries - more cost-effective SYBR green or more specific TaqMan probe. Based on this optimized method we developed an assay for detection of miR-142-5p, potential marker of colorectal cancer. We measured miR-142-5p profiles in the real samples of whole blood, plasma and serum of healthy donors. The results showed an excellent correlation with the TaqMan qPCR assay and miREIA® immuno-based technology for absolute quantification of miRNA. We conclude, that using SplintR® ligase for miRNA detection is potentially useful for clinical diagnostics. This work was funded by the Ministry of Industry and Trade of Czech Republic, project No. CZ.01.1.02/0.0/0.0/16_084/0008832. Citation Format: Iveta Hynstova, Tereza Mrackova, Michaela Adamcova, Barbora Dvorakova, Zlata Stastna, Ondrej Slaby, Milan Bartos, Viktor Ruzicka. New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-388.

MicroRNA and Diabetic Complications: A Clinical Perspective.

The rising global prevalence of diabetes and its debilitating complications give rise to significant disability and premature mortality. Due to the silent nature of diabetes and its vascular complications, and limitations in current methods for detection, there is a need for novel biomarkers for early detection and prognosis. Recent Advances: Metabolic memory and epigenetic factors are important in the pathogenesis of diabetic complications and interact with genetic variants, metabolic factors, and clinical risk factors. Micro(mi)RNAs interact with epigenetic mechanisms and pleiotropically mediate the effects of hyperglycemia on the vasculature. Utilizing mature profiling techniques and platforms, an increasing number of miRNA signatures and interaction networks have been identified for diabetes and its related cardiorenal complications. As a result, these short, single-stranded molecules are emerging as potential diagnostic and predictive tools in human studies, and may function as disease biomarkers, as well as treatment targets. However, there is complex interaction between the genome and epigenome. The regulation of miRNAs may differ across species and tissues. Most profiling studies to date lack validation, often requiring large, well-characterized cohorts and reliable normalization strategies. Furthermore, the incremental benefits of miRNAs as biomarkers, beyond prediction provided by traditional risk factors, are critical issues to consider, yet often neglected in published studies. All in all, the future for miRNA-based diagnostics and therapeutics for diabetic complications appears promising. Improved understanding of the complex mechanisms underlying miRNA dysregulation, and more well-designed studies utilizing prospective samples would facilitate the translation to clinical use.

Digital direct detection of microRNAs using single molecule arrays.

MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.

MicroRNAs as master regulators of immune responses in transplant recipients.

MicroRNAs (miRNAs) have emerged as highly evolutionarily conserved moieties that have very selective gene-regulatory functions. miRNAs are being researched for their use as potential biomarkers for diagnostics, routine prognostics as well as selective therapeutics in cancer, infectious diseases, autoimmune disorders and transplantation. This review summarizes how immune regulation by miRNAs affects the outcome of transplantation. Many miRNAs have been identified as selective markers for specific disease states and transplant conditions in the past two decades. In this review, we will discuss the recent advances and some seminal discoveries in miRNA research and their role as immune regulators in transplantation. Lastly, we will highlight the ongoing clinical trials for miRNA-based therapeutics for clinical applications and present our opinion on the future of miRNA-based diagnostics and therapeutics. miRNA-based diagnosis is a fast-moving field with new miRNA signatures being identified each day. Recent advances have also been successful at taking a few of these miRNAs to clinical trials for therapeutic interventions.

Cardio-miRNAs and onco-miRNAs: circulating miRNA-based diagnostics for non-cancerous and cancerous diseases.

Cardiovascular diseases and cancers are the leading causes of morbidity and mortality in the world. MicroRNAs (miRNAs) are short non-coding RNAs that primarily repress target mRNAs. Here, miR-24, miR-125b, miR-195, and miR-214 were selected as representative cardio-miRs that are upregulated in human heart failure. To bridge the gap between miRNA studies in cardiology and oncology, the targets and functions of these miRNAs in cardiovascular diseases and cancers will be reviewed. ACVR1B, BCL2, BIM, eNOS, FGFR3, JPH2, MEN1, MYC, p16, and ST7L are miR-24 targets that have been experimentally validated in human cells. ARID3B, BAK1, BCL2, BMPR1B, ERBB2, FGFR2, IL6R, MUC1, SITR7, Smoothened, STAT3, TET2, and TP53 are representative miR-125b targets. ACVR2A, BCL2, CCND1, E2F3, GLUT3, MYB, RAF1, VEGF, WEE1, and WNT7A are representative miR-195 targets. BCL2L2, ß-catenin, BIM, CADM1, EZH2, FGFR1, NRAS, PTEN, TP53, and TWIST1 are representative miR-214 targets. miR-125b is a good cardio-miR that protects cardiomyocytes; miR-195 is a bad cardio-miR that elicits cardiomyopathy and heart failure; miR-24 and miR-214 are bi-functional cardio-miRs. By contrast, miR-24, miR-125b, miR-195, and miR-214 function as oncogenic or tumor suppressor miRNAs in a cancer (sub)type-dependent manner. Circulating miR-24 is elevated in diabetes, breast cancer and lung cancer. Circulating miR-195 is elevated in acute myocardial infarction, breast cancer, prostate cancer and colorectal adenoma. Circulating miR-125b and miR-214 are elevated in some cancers. Cardio-miRs and onco-miRs bear some similarities in functions and circulation profiles. miRNAs regulate WNT, FGF, Hedgehog and other signaling cascades that are involved in orchestration of embryogenesis and homeostasis as well as pathogenesis of human diseases. Because circulating miRNA profiles are modulated by genetic and environmental factors and are dysregulated by genetic and epigenetic alterations in somatic cells, circulating miRNA association studies (CMASs) within several thousands of cases each for common non-cancerous diseases and major cancers are necessary for miRNA-based diagnostics.

The role of microRNAs in human breast cancer progression.

Over the past decade, microRNAs (miRNAs) have become a new paradigm of gene regulation. miRNAs are involved in a wide array of carcinogenic processes. Indeed, increasing evidence has shown the importance of miRNAs in cancer, suggesting their possible use as diagnostic, predictive and prognostic biomarkers, leading to miRNA-based anti-cancer therapies, either alone or in combination with current targeted therapies, with the goal of improving cancer treatment responses and increasing cure rates. The advantage of using a miRNA approach is based on the ability to concurrently target multiple effectors of pathways involved in cell proliferation, migration and survival. This review sheds new light on miRNA regulation of genes that play critical roles in the process of malignant transformation and tumour metastasis, the dysregulation of miRNA expression in cancer development and the development of miRNA-based diagnostics and therapeutics.