COT-3 Exosomal microRNA expression signature in blood and cerebrospinal fluid of glioblastoma patients

Analysis of exosomes derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of exosomes in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in exosomes derived from the plasma and CSF of glioblastoma patients. To this end, we first employed miRNA arrays to measure the expression of exosomal miRNAs in the plasma from glioblastoma patients (n = 24) and healthy volunteers (n = 7) as control. In addition, we performed global miRNA profiling of exosomal miRNAs in the CSF from glioblastoma patients (n = 5) and non-tumoral patients (n = 3; hydrocephalus patients) as control. In plasma derived exosomes, 80 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. In CSF, 92 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. Combined analysis of plasma and CSF revealed a similar fold difference in eight miRNAs. Next, we measured these eight miRNAs expression in in the plasma from pre- and post-operative glioblastoma patients (n = 9). Among these eight miRNAs, we identified only one miRNA (miR-34b-3p) that was upregulated in exosomes from pre-operative glioblastoma patients. Our results suggest that miR-34b-3p might have a potential as a novel diagnostic marker or a therapeutic tool for glioblastoma patients.

miRNome profiling in Duchenne Muscular Dystrophy; Identification of Asymptomatic and Manifesting Female Carriers

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder that occurs due to inactivating mutations in DMD gene, leading to muscular dystrophy. Prediction of pathological complications of DMD and the identification of female carriers are important research points that aim to reduce disease burden. Herein, we describe a case of a late DMD patient and his immediate female family members, who all carry same DMD mutation and exhibited varied degrees of symptoms. In our study, we sequenced the whole miRNome in leukocytes and plasma of the family members and results were validated using Real-Time PCR. Our results highlighted the role of miR-409-3p, miR-424-5p, miR-144-3p as microRNAs that show correlation with the extent of severity of muscular weakness and can be used for detection of asymptomatic carriers. Cellular and circulating levels of miR-494-3p had showed significant increase in symptomatic carriers, which may indicate significant roles played by this miRNA in the onset of muscular weakness. Interestingly, circulating levels of miR-206 and miR-410-3p were significantly increased only in the severely symptomatic carrier. In conclusion, our study highlighted several miRNA species, which could be used in predicting the onset of muscle and/or neurological complications in DMD carriers.

Physical Activity as a Preventive Lifestyle Intervention Acts Through Specific Exosomal miRNA Species—Evidence From Human Short- and Long-Term Pilot Studies

Exercise initiates systemic adaptation to promote health and prevent various lifestyle-related chronic diseases. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. Yet to date, a comprehensive profile of the exosomal miRNA (exomiR) content released following short-term (0.5 year in this study) and long-term (25 + years in this study) regular bouts of exercise is still lacking. However, a better understanding of these miRNA species would assist in clarifying the role of regular exercise at the molecular level in the prevention of chronic diseases. In the present pilot studies we analyzed serum exomiR expression in healthy young, sedentary participants (n = 14; age: 23 ± 2 years) at baseline and following a half year-long moderate-intensity regular exercise training. We also analyzed serum exomiR expression in older, healthy trained participants (seniors, n = 11; age: 62 ± 6 years) who engaged in endurance activities for at least 25 years. Following the isolation and enrichment of serum exosomes using Total Exosome Isolation Reagent (TEI) their exomiR levels were determined using the amplification-free Nanostring platform. Hierarchical cluster analysis revealed that the majority of exomiRs overlap for short-term (0.5 year in this study) and long-term (25 + years in this study) regular bouts of exercise. The top 12 significantly altered exomiRs (let-7a-5p; let-7g-5p; miR-130a-3p; miR-142-3p; miR-150-5p; miR-15a-5p; miR-15b-5p; miR-199a-3p; miR-199b-3p; miR-223-3p; miR-23a-3p, and miR-451a-3p) were used for further evaluation. According to KEGG pathway analysis a large portion of the exomiRs target chronic diseases including cancer, neurodegenerative and metabolic diseases, and viral infections. Our results provide evidence that exosomal miRNA modulation is the molecular mechanism through which regular exercise prevents various chronic diseases. The possibility of using such exomiRs to target diseases is of great interest. While further validation is needed, our comprehensive exomiR study presents, for the first time, the disease-preventive molecular pattern of both short and long-term regular exercise.

Transcriptome-wide identification and characterization of microRNAs in diverse phases of wood formation in Populus trichocarpa

We applied miRNA expression profiling method to Populus trichocarpa stems of the three developmental stages, primary stem (PS), transitional stem (TS), and secondary stem (SS), to investigate miRNA species and their regulation on lignocellulosic synthesis and related processes. We obtained 892, 872, and 882 known miRNAs and 1,727, 1,723, and 1,597 novel miRNAs, from PS, TS, and SS, respectively. Comparisons of these miRNA species among different developmental stages led to the identification of 114, 306, and 152 differentially expressed miRNAs (DE-miRNAs), which had 921, 2,639, and 2,042 candidate target genes (CTGs) in the three respective stages of the same order. Corelation analysis revealed 47, 439, and 71 DE-miRNA-CTG pairs of high negative correlation in PS, TS and SS, respectively. Through biological process analysis, we finally identified 34, 6, and 76 miRNA-CTG pairs from PS, TS, and SS, respectively, and the miRNA target genes in these pairs regulate or participate lignocellulosic biosynthesis related biological processes: cell division and differentiation, cell wall modification, secondary cell wall biosynthesis, lignification, and programmed cell death processes. This is the first report on an integrated analysis of genome-wide mRNA and miRNA profilings during multiple phases of poplar stem development. Our analysis results imply that individual miRNAs modulate secondary growth and lignocellulosic biosynthesis through regulating transcription factors and lignocellulosic biosynthetic pathway genes, resulting in more dynamic promotion, suppression, or regulatory circuits. This study advanced our understanding of many individual miRNAs and their essential, diversified roles in dynamic regulation of secondary growth in woody tree species.

Exosomal microRNAs as Biomarkers and Therapeutic Targets for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second most common cause of cancer-related death globally. This type of liver cancer is frequently detected at a late stage by current biomarkers because of the high clinical and biological heterogeneity of HCC tumours. From a plethora of molecules and cellular compounds, small nanoparticles with an endosomal origin are valuable cancer biomarkers or cargos for novel treatments. Despite their small sizes, in the range of 40–150 nm, these particles are delimited by a lipid bilayer membrane with a specific lipid composition and carry functional information—RNA, proteins, miRNAs, long non-coding RNAs (lncRNAs), or DNA fragments. This review summarizes the role of exosomal microRNA (miRNA) species as biomarkers in HCC therapy. After we briefly introduce the exosome biogenesis and the methods of isolation and characterization, we discuss miRNA’s correlation with the diagnosis and prognosis of HCC, either as single miRNA species, or as specific panels with greater clinical impact. We also review the role of exosomal miRNAs in the tumourigenic process and in the cell communication pathways through the delivery of cargos, including proteins or specific drugs.

Serum Exosomes and Their miRNA Load—A Potential Biomarker of Lung Cancer

Early detection of lung cancer in screening programs is a rational way to reduce mortality associated with this malignancy. Low-dose computed tomography, a diagnostic tool used in lung cancer screening, generates a relatively large number of false-positive results, and its complementation with molecular biomarkers would greatly improve the effectiveness of such programs. Several biomarkers of lung cancer based on different components of blood, including miRNA signatures, were proposed. However, only a few of them have been positively validated in the context of early cancer detection yet, which imposes a constant need for new biomarker candidates. An emerging source of cancer biomarkers are exosomes and other types of extracellular vesicles circulating in body fluids. Hence, different molecular components of serum/plasma-derived exosomes were tested and showed different levels in lung cancer patients and healthy individuals. Several studies focused on the miRNA component of these vesicles. Proposed signatures of exosome miRNA had promising diagnostic value, though none of them have yet been clinically validated. These signatures involved a few dozen miRNA species overall, including a few species that recurred in different signatures. It is worth noting that all these miRNA species have cancer-related functions and have been associated with lung cancer progression. Moreover, a few of them, including known oncomirs miR-17, miR-19, miR-21, and miR-221, appeared in multiple miRNA signatures of lung cancer based on both the whole serum/plasma and serum/plasma-derived exosomes.

Human Cytomegalovirus-Encoded microRNAs Can Be Found in Saliva Samples from Renal Transplant Recipients

Human cytomegalovirus (HCMV) infections are common following renal transplantation and may have long-lasting effects. HCMV can be measured directly by viral DNA or indirectly via host immune responses. HCMV-encoded microRNA (miRNA) may alter the pathobiology of HCMV infections and contribute to the progression of HCMV disease. HCMV-encoded miRNAs can be detected in blood but have not been sought in saliva. We investigated saliva samples from 32 renal transplant recipients (RTR) and 12 seropositive healthy controls for whom immunological data was available. Five HCMV-encoded miRNAs (miR-UL112-5p, miR-US5-2-3p, miR-UL36, miR-US25-2-3p and miR-UL22A) were sought using primer probe assays. HCMV miRNA species were detected in saliva from 15 RTR and 3 healthy controls, with miR-US5-2-3p most commonly detected. The presence of HCMV miRNAs associated with increased T-cell responses to HCMV IE-1 in RTR, suggesting a link with frequent reactivations of HCMV.

Premature MicroRNA-Based Therapeutic: A “One-Two Punch” against Cancers

Up-to-date knowledge regarding the biogenesis and functioning of microRNAs (miRNAs) has provided a much more comprehensive and concrete view of miRNA biology than anyone ever expected. Diverse genetic origins and biogenesis pathways leading to functional miRNAs converge on the synthesis of ≈21-nucleotide RNA duplex, almost all of which are processed from long premature sequences in a DICER- and/or DROSHA-dependent manner. Formerly, it was assumed that one mature strand of the duplex is preferentially selected for entry into the silencing complex, and the paired passenger strands (miRNA*) are subjected to degradation. However, given the consolidated evidence of substantial regulatory activity of miRNA* species, currently, this preconception has been overturned. Here, we see the caveat and opportunity toward exogenously manipulating the expression of premature miRNA, leading to simultaneous upregulation or downregulation of dual regulatory strands due to altered expressions. The caveat is the overlooked miRNA* interference while manipulating the expression of a target miRNA at the premature stage, wherein lies the opportunity. If the dual strands of a pre-miRNA function synergistically, the overlooked miRNA* interference may inversely optimize the therapeutic performance. Insightfully, targeting the premature miRNAs may serve as the “one-two punch” against diseases, especially cancers, and this has been discussed in detail in this review.

The abundance of a transfer RNA-derived RNA fragment small RNA subpopulation is enriched in cauda spermatozoa

The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis; a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.