MicroRNAs and Cardiovascular Disease Risk.

PURPOSE OF REVIEW: MicroRNAs (miRNAs)-short, non-coding RNAs-play important roles in almost all aspects of cardiovascular biology, and changes in intracellular miRNA expression are indicative of cardiovascular disease development and progression. Extracellular miRNAs, which are easily measured in blood and can be reflective of changes in intracellular miRNA levels, have emerged as potential non-invasive biomarkers for disease. This review summarizes current knowledge regarding miRNAs as biomarkers for assessing cardiovascular disease risk and prognosis. RECENT FINDINGS: Numerous studies over the last 10-15 years have identified associations between extracellular miRNA profiles and cardiovascular disease, supporting the potential use of extracellular miRNAs as biomarkers for risk stratification. However, clinical application of extracellular miRNA profiles has been hampered by poor reproducibility and inter-study variability that is due largely to methodological differences between studies. While recent studies indicate that circulating extracellular miRNAs are promising biomarkers for cardiovascular disease, evidence for clinical implementation is lacking. This highlights the need for larger, well-designed studies that use standardized methods for sample preparation, miRNA isolation, quantification, and normalization.

Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides.

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

MicroRNAs in Cancer: From Gene Expression Regulation to the Metastatic Niche Reprogramming.

By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors' opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.

VAMP3 and SNAP23 mediate the disturbed flow-induced endothelial microRNA secretion and smooth muscle hyperplasia.

Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication.

Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics.

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.

MicroRNAs as Potential Biomarkers in Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive type of skin cancer associated with a poor prognosis. This carcinoma was named after its presumed cell of origin, the Merkel cell, which is a mechanoreceptor cell located in the basal epidermal layer of the skin. Merkel cell polyomavirus seems to be the major causal factor for MCC because approximately 80% of all MCCs are positive for viral DNAs. UV exposure is the predominant etiological factor for virus-negative MCCs. Intracellular microRNA analysis between virus-positive and virus-negative MCC cell lines and tumor samples have identified differentially expressed microRNAs. Comparative microRNA profiling has also been performed between MCCs and other non-MCC tumors, but not between normal Merkel cells and malignant Merkel cells. Finally, Merkel cell polyomavirus encodes one microRNA, but its expression in virus-positive MCCs is low, or non-detectable or absent, jeopardizing its biological relevance in tumorigenesis. Here, we review the results of microRNA studies in MCCs and discuss the potential application of microRNAs as biomarkers for the diagnosis, progression and prognosis, and treatment of MCC.

Lipoprotein carriers of microRNAs.

Lipoproteins, namely high-density lipoproteins (HDL), transport a wide-variety of cargo in addition to cholesterol and lipids. In 2011, HDL and low-density lipoproteins (LDL) were reported to transport microRNAs (miRNA). Since the original discovery, there has been great excitement for this topic and a handful of follow-up publications. Here, we review the current landscape of lipoprotein transport of miRNAs. HDL-miRNAs have been demonstrated to be altered in cardiovascular disease (CVD), including hypercholesterolemia and atherosclerosis. As such, HDL- and LDL-miRNAs may represent a novel class of disease biomarkers. Below, we review HDL-miR-92a and miR-486 levels in myocardial infarction and unstable angina, and HDL-miR-223 and miR-24 levels in coronary artery disease (CAD). Moreover, we address HDL's contribution to the total pool of extracellular miRNAs in plasma and differential distribution of miRNAs across HDL subspecies. Finally, we address current and future challenges for this new field and the barriers to such work. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.

Identifying the Signatures and Rules of Circulating Extracellular MicroRNA for Distinguishing Cancer Subtypes.

Cancer is one of the most threatening diseases to humans. It can invade multiple significant organs, including lung, liver, stomach, pancreas, and even brain. The identification of cancer biomarkers is one of the most significant components of cancer studies as the foundation of clinical cancer diagnosis and related drug development. During the large-scale screening for cancer prevention and early diagnosis, obtaining cancer-related tissues is impossible. Thus, the identification of cancer-associated circulating biomarkers from liquid biopsy targeting has been proposed and has become the most important direction for research on clinical cancer diagnosis. Here, we analyzed pan-cancer extracellular microRNA profiles by using multiple machine-learning models. The extracellular microRNA profiles on 11 cancer types and non-cancer were first analyzed by Boruta to extract important microRNAs. Selected microRNAs were then evaluated by the Max-Relevance and Min-Redundancy feature selection method, resulting in a feature list, which were fed into the incremental feature selection method to identify candidate circulating extracellular microRNA for cancer recognition and classification. A series of quantitative classification rules was also established for such cancer classification, thereby providing a solid research foundation for further biomarker exploration and functional analyses of tumorigenesis at the level of circulating extracellular microRNA.

Salivary exosomal microRNAs as biomarkers for head and neck cancer detection-a literature review.

BACKGROUND: MicroRNAs (miRs) are small, non-coding mRNA molecules which regulate cellular processes in tumorigenesis. miRs were discovered in extracellular environment and biological fluids, carrying marks of head and neck squamous cell carcinoma (HNSCC). They were also identified in abundance in salivary exosomes, in which they are protected by exosome lipid barrier against enzymatic injuries and therefore, the accuracy of exosomal miR-based cancer detection increase. This systematic review aimed to reveal and inventorize the most reliable exosomal miRNAs in saliva samples which can be used as novel biomarkers for early detection of HNSCC. MATERIALS AND METHODS: A systematic literature search, according to PRISMA guideline, was performed on Pubmed and Google Academic libraries, based on specific keywords. Original articles published between 2010 and 2021 were selected. The quality of each paper was assessed using the Quality Evaluation Scoring Tool. RESULTS: At the end of selection process, five studies met the inclusion criteria. These studies analyzed twelve salivary exosomal miRs, presenting different methods of exosome and miR identification for HNSCC detection. A comprehensive explanation of the miR pathways of action was drawn and illustrated in this review. CONCLUSION: Exosomal miRs are promising biomarkers for oral cavity and oropharyngeal cancer detection. miR-10b-5p, miR-486-5p, miR-24-3p and miR-200a stand as the most useful ones in saliva sample examination.

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells.

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210-3p, and miR-224-5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210-3p or miR-224-5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210-3p, and miR-224-5p in sEVs was compared. The amount of miR-33a-5p and miR-210-3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224-5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210-3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.

Circulating microRNAs as biomarkers in cancer diagnosis.

MicroRNAs (miRNAs) are a group of tiny molecules of 18-22 nucleotide long noncoding RNA that regulate the post-transcriptional gene expression through translational inhibition and/or mRNA destabilization. Because of their involvement in important developmental processes, it is highly likely that the altered expression of miRNAs could be associated with abnormal conditions like suboptimal growth or diseases. Thus, the expression of miRNAs can be used as biomarkers in pathophysiological conditions. Recently, a handful of miRNAs are detected in cell-free conditions including biofluids and cell culture media and they exhibit specific expression patterns that are associated with altered physiological conditions. Extracellular miRNAs are not only extremely stable outside cells in a variety of biofluids but also they are easy to acquire. These characteristics led to the idea of using extracellular miRNAs as a potential biomarker for the onset and prognosis of cancer. Although miRNAs have been proposed as a potential diagnostic tool for cancer detection, their application in the routine clinical investigation is yet to come. First, this review will provide an insight into the extracellular miRNAs, particularly, their release mechanisms and characteristics, and the potential of extracellular miRNAs as a biomarker in cancer detection. Finally, it will discuss the potential of using extracellular miRNAs in different cancer diagnoses and challenges associated with the clinical application of extracellular miRNAs as noninvasive biomarkers.

The diagnostic, prognostic and therapeutic potential of circulating microRNAs in ovarian cancer.

Ovarian cancer (OC) is often diagnosed at an advanced stage because of the late onset of symptoms, and this together with the lack of effective treatments, has meant it is associated with a very high mortality. The aberrant expression of MicroRNA (miRNA) contributes to the initiation and development of human tumors including OC. Several miRNAs are secreted by tumor cells and can be identified in body fluids. Serum miRNAs levels are associated with several clinical conditions, and may be used to predict prognosis and response to treatments in some cancers including OC. This review summarizes the current progresses regarding the potential applications of circulating miRNA as innovative biomarkers in OC.

Liquid profiling in plants: identification and analysis of extracellular metabolites and miRNAs in pollination drops of Ginkgo biloba.

The pollination drop (PD), also known as an ovular secretion, is a critical feature of most wind-pollinated gymnosperms and function as an essential component of pollination systems. However, the metabolome and small RNAs of gymnosperm PDs are largely unknown. We employed gas chromatography-mass spectrometry to identify a total of 101 metabolites in Ginkgo biloba L. PDs. The most abundant metabolites were sugars (45.70%), followed by organic acids (15.94%) and alcohols (15.39%) involved in carbohydrate metabolism, glycine, serine and threonine metabolism. Through pollen culture of the PDs, we further demonstrated that the metabolic components of PDs are indispensable for pollen germination and growth; in particular, organic acids and fatty acids play defensive roles against microbial activity. In addition, we successfully constructed a small RNA library and detected 45 known and 550 novel miRNAs in G. biloba PDs. Interestingly, in a comparative analysis of miRNA expression between PDs and ovules, we found that most of the known miRNAs identified in PDs were also expressed in the ovules, implying that miRNAs in PDs may originate from ovules. Further, combining with potential target prediction, degradome validation and transcriptome sequencing, we identified that the interactions of several known miRNAs and their targets in PDs are involved in carbohydrate metabolism, hormone signaling and defense response pathways, consistent with the metabolomics results. Our results broaden the knowledge of metabolite profiling and potential functional roles in gymnosperm PDs and provide the first evidence of extracellular miRNA functions in ovular secretions from gymnosperms.

Extracellular microRNAs in blood differentiate between ischaemic and haemorrhagic stroke subtypes.

Rapid identification of patients suffering from cerebral ischaemia, while excluding intracerebral haemorrhage, can assist with patient triage and expand patient access to chemical and mechanical revascularization. We sought to identify blood-based, extracellular microRNAs 15 (ex-miRNAs) derived from extracellular vesicles associated with major stroke subtypes using clinical samples from subjects with spontaneous intraparenchymal haemorrhage (IPH), aneurysmal subarachnoid haemorrhage (SAH) and ischaemic stroke due to cerebral vessel occlusion. We collected blood from patients presenting with IPH (n = 19), SAH (n = 17) and ischaemic stroke (n = 21). We isolated extracellular vesicles from plasma, extracted RNA cargo, 20 sequenced the small RNAs and performed bioinformatic analyses to identify ex-miRNA biomarkers predictive of the stroke subtypes. Sixty-seven miRNAs were significantly variant across the stroke subtypes. A subset of exmiRNAs differed between haemorrhagic and ischaemic strokes, and LASSO analysis could distinguish SAH from the other subtypes with an accuracy of 0.972 ± 0.002. Further analyses predicted 25 miRNA classifiers that stratify IPH from ischaemic stroke with an accuracy of 0.811 ± 0.004 and distinguish haemorrhagic from ischaemic stroke with an accuracy of 0.813 ± 0.003. Blood-based, ex-miRNAs have predictive value, and could be capable of distinguishing between major stroke subtypes with refinement and validation. Such a biomarker could one day aid in the triage of patients to expand the pool eligible for effective treatment.

Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation.

MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3' untranslated region (3' UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5' UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication. In this review, we provide an update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations. We also summarize the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs.

Extracellular microRNAs in Membrane Vesicles and Non-vesicular Carriers.

Great excitement has surrounded the finding that small RNAs are stable in various biofluids and carry specific signatures reflecting physiological and pathological states. In this chapter, we briefly describe the impact of this revolutionary discovery and introduce different subclasses of circulating microRNAs based on their mode of transport. Subsequently, we review the current state-of-the art knowledge on microRNA selection for export, secretion and possible uptake mechanisms and their potential function in circulation. Furthermore, we give an overview on the possible use of cell-free microRNAs as biomarkers and as therapeutic targets. Overall, we aim to highlight open questions and address some of the pitfalls of current extracellular RNA research.

Extracellular microRNAs in bronchoalveolar lavage samples from patients with lung diseases as predictors for lung cancer.

OBJECTIVE: The detection of tumor-associated microRNA expression in bronchial lavage (BL) samples of lung cancer patients could improve the non-invasive tumor diagnostic. METHODS: The profile of extracellular microRNAs in bronchial lavage was evaluated using three pools for lung cancer group (malignant) and three pools for non-cancerous group (benign) of 10 patients each. To confirm the results for the selected microRNAs in a qRT-PCR the BL of 30 different lung cancer and non-cancerous patients was used. We examined total-RNA from cell-free supernatant of BL samples. For normalization we added exogenous cel-miR 39. RESULTS: Using microRNA arrays we found a panel of eight microRNAs (hsa-miR 19b-1, 1285, 1289, 1303, 217, 29a-5p, 548-3p, 650) that were differentially expressed between the lung cancer and the non-cancerous group. Further investigation by qPCR revealed five microRNAs (U6 snRNA, hsa-miR 1285, 1303, 29a-5p, 650) that were significantly up-regulated in patients with lung cancer. CONCLUSIONS: In bronchial lavage samples the five microRNAs identified in this study have a diagnostic potential for use as noninvasive biomarkers in lung cancer.

Exosome and exosomal microRNA: trafficking, sorting, and function.

Exosomes are 40-100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sorting mechanism of exosomal miRNAs, since the miRNA profiles of exosomes may differ from those of the parent cells. Exosomal miRNAs play an important role in disease progression, and can stimulate angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exosomal miRNAs, and briefly describe how exosomes and their miRNAs function in recipient cells. Finally, we will discuss the potential applications of these miRNA-containing vesicles in clinical settings.

Effective detection and quantification of dietetically absorbed plant microRNAs in human plasma.

The detection of exogenous plant microRNAs in human/animal plasma/sera lies at the foundation of exploring their cross-kingdom regulatory functions. It is necessary to establish a standard operation procedure to promote study in this nascent field. In this study, 18 plant miRNAs were assessed in watermelon juice and mixed fruits by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CT values, no-template controls and standard curves for each miRNA were used to evaluate the specificity and sensitivity of qRT-PCR and to obtain concentrations. Sixteen miRNAs were selected and measured in human plasma from volunteers after drinking juice. The CT values of 6 plant miRNAs in human plasma fell outside the linear ranges of their standard curves. The remaining 10 miRNAs were present at high basal levels, and 6 of them showed a dynamic physiological pattern in plasma (absorption rates of 0.04% to 1.31%). Northern blotting was used to confirm the qRT-PCR results. Critical issues such as RNA extraction and internal controls were also addressed.