Extracellular miRNA biomarkers in neurologic disease: is cerebrospinal fluid helpful?

Aim: The aim of our work is to aggregate data from publications of cerebrospinal fluid extracellular miRNA to identify candidate diagnostic biomarkers, and those warranting further study. Materials & methods: Data were pooled from nine studies, encompassing 864 patients across 16 diseases. Unsupervised clustering grouped patients by a broad category of diseases. Results & conclusion: Compared with healthy controls, in patients with Alzheimer’s disease, hsa-miR-767-5p was overexpressed (p < 0.001) and in patients with Huntington’s disease, hsa-miR-361-3p was underexpressed (p < 10-4). We also define a subset of extracellular miRNA as candidate biomarkers that are robustly detected across patients, studies and diseases; thereby, warranting further study.

Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes

Extracellular circulating microRNAs (miRNAs) are currently a focus of interest as non-invasive biomarkers of cardiovascular pathologies, including coronary artery disease (CAD) and acute coronary syndromes (ACS): myocardial infarction with and without ST-segment elevation (STEMI and NSTEMI) and unstable angina (UA). However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological variances in different studies. In this work, we fulfilled the basic pre-analytical requirements provided for circulating miRNA studies for application to stable CAD and ACS research. We used quantitative PCR to determine the relative plasma levels of eight circulating miRNAs that are potentially associated with atherosclerosis. In a cohort of 136 adult clinic CAD patients and outpatient controls, we found that the plasma levels of miR-21-5p and miR-146a-5p were significantly elevated in ACS patients, and the level of miR-17-5p was decreased in ACS and stable CAD patients compared to both healthy controls and hypertensive patients without CAD. Within the ACS patient group, no differences were found in the plasma levels of these miRNAs between patients with positive and negative troponin, nor were any differences found between STEMI and NSTEMI. Our results indicate that increased plasma levels of miR-146a-5p and miR-21-5p can be considered general ACS circulating biomarkers and that lowered miR-17-5p can be considered a general biomarker of CAD.

The itinerary of circulating miRNAs – implications for cancer progression and diagnosis

microRNAs (miRNAs) are non-coding RNAs that regulate gene expression and protect cells from foreign nucleic acids. miRNA is produced in the nucleus and processed in the cytoplasm. These small nucleic acid molecules are released from cells to the extracellular matrix (extracellular miRNA, ex-miRNA) and reach blood plasma (circulating miRNA). Circulating miRNA can also be detected in other biological fluids, such as saliva, cerebrospinal fluid or urine, and it is usually carried by proteins or extracellular vesicles. Argonaute-miRNA, or miRNA-lipoprotein complex, protect miRNA from being degraded. The entrance of extracellular miRNA into a target cell is mediated by endocytosis and membrane fusion of extracellular vesicles. Additionally, miRNA can also be delivered in high-density lipoproteins by means of interactions with scavenger receptors. miRNAs absorbed into a cell can act as tumour promoters (oncomirs), or suppressors by inhibiting the translation process of the target mRNAs, thus, affecting cells in the tumour microenvironment. miRNA can impact other cells by supporting tumour growth, promoting angiogenesis and modulating the immune system. Molecular high-throughput methods are employed to detect circulating miRNA, and a potentially helpful diagnostic test has been designed to characterise the cancer type. In this review, we aim to summarise the itinerary of miRNAs from a source cell to a target cell, as well as to show how this class of small nucleic acids participates in intercellular communication. Finally, we highlight examples of miRNAs usage as potential molecular markers and discuss treatment approaches in clinical trials.

Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2 = 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72–16.95 copies/oocyte), Hprt1 (1.05–19.05 copies/oocyte) and ATPase 6, (5.55–32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic–uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription–quantitative polymerase chain reaction–based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.

Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion

ABSTRACTExtracellular environment consists of a plethora of different molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, it is generally accepted that most of the biological activity is attributed to EV-associated miRNAs. The capability of EVs to transport cargoes has attracted much interest towards developing EVs as therapeutic short RNA carriers by using endogenous loading strategies for miRNA enrichment. Here, by overexpressing miRNA and shRNA sequences of interest in source cells and using size exclusion liquid chromatography (SEC) to separate the cellular secretome into EV and non-EV fractions, we saw that strikingly, <2% of all secreted overexpressed miRNA were found in association with EVs. To see whether the prominent non-EV miRNA secretion also holds true at the basal expression level of native miRNA transcripts, both fractions were further analysed by small RNA sequencing. This revealed a global correlation of EV and non-EV miRNA abundance to that of their parent cells and showed an enrichment only for miRNAs with a relatively low cellular expression level. Further quantification showed that similarly to the transient overexpression context, an outstanding 96.2-99.9% of total secreted miRNA at its basal level was secreted to the non-EV fraction. Yet, even though EVs contain only a fraction of secreted miRNAs, these molecules were found stable at 37°C in serum-containing environment, indicating that if sufficient miRNA loading to EVs is achieved, EVs can remain miRNA delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy can be a relatively wasteful way of loading miRNA to EVs and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

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.