miRNA Annotation Research Articles

The microRNAome of Strongylus vulgaris larvae and their excretory/secretory products with identification of parasite-derived microRNAs in horse arterial tissue

The equine bloodworm, Strongylus vulgaris, is a highly pathogenic parasite causing potentially fatal vascular and intestinal damage. Parasites express and release microRNAs (miRNAs) for internal regulation and to modulate host immunity. The complete set of miRNAs expressed by S. vulgaris (the S. vulgaris miRNAome) remains unannotated and the aim of this study was to annotate the miRNAome of L4 and L5 stages of S. vulgaris, and to examine differences in miRNA abundance between larval stages and sexes. Furthermore, we aimed to determine if miRNAs were detectable in excretory/secretory products (ESPs) from larvae and in arterial tissue from their predilection site, the cranial mesenteric artery (CMA). Larvae were collected from naturally infected foals, and categorized by sex and stage. A subset of larvae was snap-frozen, while those remaining were incubated and the (ESPs) collected. Arterial tissue samples were collected from the CMA. Small RNA sequencing, followed by a custom bioinformatic pipeline, was used for annotation. We identified 142 S. vulgaris miRNAs in larvae and 136 in ESPs. Significant differences in miRNA abundance were observed between larvae and ESPs, and between L5 females (L5Fs) and L5 males (L5Ms), L4s and L5Fs, and L4s and L5Ms. No differences were found between L4s and L5s overall. In ESPs, several miRNAs were differentially abundant across all groups. Validation through quantitative real-time PCR (qPCR) detected selected miRNAs and their differential abundance in larvae and ESPs. One parasite-derived miRNA was detected in some of the horse arterial tissue samples but at very low levels. This study provided the first annotation of the S. vulgaris miRNAome. Most of the annotated larval miRNAs were also detectable in ESPs, and differences in miRNA abundance between sexes were found for larvae, and between sexes and stages for ESPs. Parasite-derived miRNAs were, however, not consistently detectable in the surrounding host arterial tissue.

Altered microRNA composition in the uterine lumen fluid in cattle (Bos taurus) pregnancies initiated by artificial insemination or transfer of an in vitro produced embryo

BackgroundMicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo.ResultsThe abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets.ConclusionsThe results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle.

MiRNA expression signatures induced by pasteurella multocida infection in goats lung

MicroRNAs (miRNAs) are important regulators of gene expression and are involved in bacterial pathogenesis and host–pathogen interactions. In this study, we investigated the function of miRNAs in the regulation of host responses to Pasteurella multocida infection. Using next-generation sequencing, we analyzed miRNA expression pattern and identified differentially expressed miRNAs in Pasteurella multocida-infected goat lungs. In addition, we investigated the function of differentially expressed miRNAs andtheir targeted signaling pathways in bacterial infection processes. The results showed that Pasteurella multocida infection led to 69 significantly differentially expressed miRNAs, including 28 known annotated miRNAs with miR-497-3p showing the most significant difference. Gene target prediction and functional enrichment analyses showed that the target genes were mainly involved in cell proliferation, regulation of the cellular metabolic process, positive regulation of cellular process, cellular senescence, PI3K-Akt signaling pathway, FoxO signaling pathway and infection-related pathways. In conclusion, these data provide a new perspective on the roles of miRNAs in Pasteurella multocida infection.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

Abstract 2272: Unlocking the potential of miRNA sequencing for early detection and monitoring of pancreatic cancer progression and recurrence

Abstract Pancreatic cancer (PC) is an elusive and deadly disease. Many patients with PC are diagnosed with metastatic or locally advanced tumors, limiting surgical resection, and increasing the risk of relapse after treatment. Relapse is recorded to be as high as 75% in the first 2 years after clinical intervention, resulting in a mean 5-year survival of 12% in the US, and as low as 2% worldwide. It is vital to develop diagnostic and monitoring techniques for PC. MicroRNAs (miRNAs) are robustly expressed in patient tumors and provide a rich source of biomarkers for disease detection and status. miRNA are short nucleic acid strands that bind to catalytic proteins to guide the regulation of large RNAs within the cellular space. This system is altered in tumors, leading to abhorrent expression of miRNA. Due to significant advancements in sequencing technology, miRNA-seq has become more accurate and affordable in detecting profile changes. To identify biomarkers for PC progression, we developed from the Gemcitabine-sensitive MIA-PaCa-2 (MP2) cell line the Gemcitabine and FOLFIRINOX resistant MP2-GR cell line. MiRNA-seq was conducted on the parental MP2 and resistant MP2-GR cells to identify those miRNAs that were differentially expressed between the cell lines. These miRNAs were analyzed via pathway analysis and gene ontology to find predicted functions followed by annotations from the miRNA Enrichment Analysis and Annotation tool (miEAA) to identify differentially expressed miRNAs associated with PC progression. This comparison indicated several miRNAs related to PC resistance. This dataset was then compared to The Cancer Genome Atlas (TCGA), which contains data from over 300 PC patients. The miRNA set was used to predict both PC progression after treatment, and recurrence. We are currently refining this data set to increase its predictive sensitivity and widen our patient data set comparison to further elucidate this approach in a clinical setting. Citation Format: Ryan N. Fuller, Ann Morcos, Desirae Escalera, Nathan R. Wall. Unlocking the potential of miRNA sequencing for early detection and monitoring of pancreatic cancer progression and recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2272.

MicroRNAs as biomarkers of brain injury in neonatal encephalopathy: an observational cohort study

Neonatal Encephalopathy (NE) is a major cause of lifelong disability and neurological complications in affected infants. Identifying novel diagnostic biomarkers in this population may assist in predicting MRI injury and differentiate neonates with NE from those with low-cord pH or healthy neonates and may help clinicians make real-time decisions. To compare the microRNA (miRNA) profiles between neonates with NE, healthy controls, and neonates with low cord pH. Moreover, miRNA concentrations were compared to brain injury severity in neonates with NE. This is a retrospective analysis of miRNA profiles from select samples in the biorepository and data registry at the University of Florida Health Gainesville. The Firefly miRNA assay was used to screen a total of 65 neurological miRNA targets in neonates with NE (n = 36), low cord pH (n = 18) and healthy controls (n = 37). Multivariate statistical techniques, including principal component analysis and orthogonal partial least squares discriminant analysis, and miRNA Enrichment Analysis and Annotation were used to identify miRNA markers and their pathobiological relevance. A set of 10 highly influential miRNAs were identified, which were significantly upregulated in the NE group compared to healthy controls. Of these, miR-323a-3p and mir-30e-5p displayed the highest fold change in expression levels. Moreover, miR-34c-5p, miR-491-5p, and miR-346 were significantly higher in the NE group compared to the low cord pH group. Furthermore, several miRNAs were identified that can differentiate between no/mild and moderate/severe injury in the NE group as measured by MRI. MiRNAs represent promising diagnostic and prognostic tools for improving the management of NE.

Association of microRNA Polymorphisms with Toxicities Induced by Methotrexate in Children with Acute Lymphoblastic Leukemia.

Methotrexate (MTX), a structurally related substance to folic acid, is an important chemotherapeutic agent used for decades in the treatment of pediatric acute lymphoblastic leukemia (ALL) and other types of cancer as non-Hodgkin lymphomas and osteosarcomas. Despite the successful outcomes observed, the primary drawback is the variability in the pharmacokinetics and pharmacodynamics between patients. The main adverse events related to its use are nephrotoxicity, mucositis, and myelosuppression, especially when used in high doses. The potential adverse reactions and toxicities associated with MTX are a cause for concern and may lead to dose reduction or treatment interruption. Genetic variants in MTX transport genes have been linked to toxicity. Pharmacogenetic studies conducted in the past focused on single nucleotide polymorphisms (SNPs) in the coding and 5'-regulatory regions of genes. Recent studies have demonstrated a significant role of microRNAs (miRNAs) in the transport and metabolism of drugs and in the regulation of target genes. In the last few years, the number of annotated miRNAs has continually risen, in addition to the studies of miRNA polymorphisms and MTX toxicity. Therefore, the objective of the present study is to investigate the role of miRNA variants related to MTX adverse effects.

Clinical and biological significance of circulating miRNAs in chronic pancreatitis patients undergoing total pancreatectomy with islet autotransplantation.

Specific microRNAs (miRNAs) were elevated in chronic pancreatitis (CP) patients during islet infusion after total pancreatectomy (TPIAT). We aimed to identify circulating miRNA signatures of pancreatic damage, predict miRNA-mRNA networks to identify potential links to CP pathogenesis and identify islet isolation and transplantation functional outcomes. Small RNA sequencing was performed to identify distinct circulating miRNA signatures in CP. Plasma miRNAs were measured using miRCURY LNA SYBR green quantitative real-time polymerase chain reaction assays. Correlation analyses were performed using R software. The miRNA target and disease interactions were determined using miRNet and the miRNA enrichment and annotation tool. Alterations were found in circulating miRNAs in CP patients compared to healthy controls. Further studies were conducted on 12 circulating miRNAs enriched in the pancreas, other tissues and other diseases including cancer and fibrosis. Approximately 2888 mRNAs in the pancreas were their targets, demonstrating interactions with 76 small molecules. Three miRNAs exhibited interactions with morphine and five exhibited interactions with glucose. The miRNA panel targeted 22 genes associated with pancreatitis. The islet-specific, acinar cell-specific and liver-specific miRNAs were elevated at 6h after islet infusion and returned to baseline levels 3 months after TPIAT. Circulating levels of miRNAs returned to pre-transplant levels 1-year post-transplant. Circulating miRNAs measured before and 6h after islet infusion were directly or inversely associated with metabolic outcomes at 3 and 6 months post-transplant. miRNAs may contribute to CP pathogenesis, and elevated circulating levels may be specific to pancreatic inflammation and fibrosis, warranting further investigation.

P07.11.B A NOVEL CRISPR/CAS9 KNOCKOUT LIBRARY UNCOVERS PAN-ESSENTIAL MIRNAS IN HUMAN CANCER CELL LINES

Abstract BACKGROUND MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play an important role during cancer initiation and progression. Genome-wide functional screening using the CRISPR/Cas9 system is a powerful tool to uncover genetic dependencies for distinct tumor entities, and has revealed novel insights into pan-cancer fitness genes. Current libraries are focused on single guide RNAs (sgRNAs) targeting protein-coding genes, thereby limiting functional genomics-based investigations of miRNA function. MATERIAL AND METHODS We here designed a novel CRISPR/Cas9 knockout library of 8,107 distinct sgRNAs targeting a total of 1,769 human miRNAs. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Screen hits per cell line were scored on the basis of a combination of a supervised Bayesian gene essentiality analysis (BAGEL2) and an unsupervised gene ranking algorithm (MAGeCK). miRNA genes that scored across all cell lines, i.e. potentially pan-essential miRNAs, were identified using a combination of the Adaptive Daisy Model (ADaM) algorithm and the Fitness Percentile (FiPer) method. RESULTS In silico analyses revealed that our library has significantly lower off-target activity for protein-coding genes as compared to previously described miRNA-targeting sgRNA libraries, while providing a higher coverage of miRNAs present in the human genome. In total, only 13% (n=230) of all targeted miRNAs induced a fitness effect on one or more cell lines, and the majority (83%) of these genes induced a dependency in less than 50% of the tested cell lines. Pan-cancer fitness gene detection by ADaM or FiPer differed solely on the level of stringency. As such, ADaM and FiPer identified 34 core fitness or 54 common essential miRNAs, respectively, and almost all core fitness genes (97%) were identified as common essential. Preliminary network analyses revealed that protein-coding genes that are regulated by core fitness miRNAs are significantly enriched for cell cycle and p53 signaling networks, providing a first rationale for their dependency profiles in human cancer cell lines. CONCLUSION We here established a novel method for functional investigation of miRNAs by means of genome-wide CRISPR/Cas9 knockout screens. Our sgRNA library outperforms previously established libraries in terms of specificity and overall miRNA coverage, making it a novel tool to investigate miRNA function in a genome-wide fashion. We here use this library to define a first-of-its-kind genome-wide functional annotation of miRNAs, revealing a subset of pan-cancer fitness miRNAs that might regulate essential cellular processes such as cell cycle progression. Our data highlight the wide potential of this library in investigating miRNA function in diverse biological settings.

Two microRNAs of plasma-derived small extracellular vesicles as biomarkers for metastatic non-small cell lung cancer

BackgroundMicroRNAs (miRNAs) of plasma-derived small extracellular vesicles (sEVs) have been proven to be associated with metastasis in several types of cancer. This study aimed to detect miRNAs of plasma-derived sEVs as potential biomarkers for metastatic non-small cell lung cancer (NSCLC).MethodsWe assessed the miRNA profiles of plasma-derived sEVs from healthy individuals as the control group (CT group), NSCLC patients without distant organ metastasis as the NM-NSCLC group and patients with distant organ metastasis as the M-NSCLC group. Next-generation sequencing (NGS) was performed on samples, and differentially expressed miRNAs (DEMs) of the three groups were screened. Kyoto Encyclopedia of Genes and Genomes (KEGG) and ClueGO were used to predict potential pathways of DEMs. MiRNA enrichment analysis and annotation tool (miEAA) was used to understand changes in the tumour microenvironment in NSCLC. Quantitative reverse transcription polymerase chain reaction (qRT‒PCR) analysis was used to validate target miRNAs.ResultNGS was performed on 38 samples of miRNAs of plasma-derived sEVs, and DEMs were screened out between the above three groups. Regarding the distribution of DEMs in the NM-NSCLC and M-NSCLC groups, KEGG pathway analysis showed enrichment in focal adhesion and gap junctions and ClueGO in the Rap1 and Hippo signaling pathways; miEAA found that fibroblasts were over-represented. From our screening, miRNA-200c-3p and miRNA-4429 were found to be predictive DEMs among the CT, NM-NSCLC and M-NSCLC groups, and qRT‒PCR was applied to verify the results. Finally, it was revealed that expression levels of miR-200c-3p and miR-4429 were significantly upregulated in M-NSCLC patients.ConclusionThis study identified miRNA-200c-3p and miRNA-4429 as potential biomarkers for NSCLC metastasis.

MicroRNA Expression Dynamics in Culicoides sonorensis Biting Midges Following Blood-Feeding.

Culicoides sonorensis midges vector multiple livestock arboviruses, resulting in significant economic losses worldwide. Due to the tight association between virus transmission, blood feeding, and egg development, understanding midge physiology is paramount to limiting pathogen transmission. Previous studies have demonstrated the importance of small non-coding RNAs (ncRNAs), specifically microRNAs (miRNAs), in multiple aspects of vector physiology. These small ncRNAs regulate gene expression at the post-transcriptional level and display differential expression during pathogen infection. Due to the lack of annotated miRNAs in the biting midge and associated expression profiles, we used small RNA-Seq and miRDeep2 analyses to determine the Culicoides miRNAs in whole females and midgut tissues in response to blood feeding. Our analyses revealed 76 miRNAs within C. sonorensis composed of 73 orthologous and three candidate novel miRNAs, as well as conserved miRNA clusters. miRNA conservation suggests an interesting evolutionary relationship between miRNA expression and hematophagy in the infraorder Culicomorpha. We also identified multiple blood meal-regulated and tissue-enriched miRNAs. Lastly, we further identified miRNAs with expression patterns potentially associated with virus infection by probing publicly available datasets. Together, our data provide a foundation for future ncRNA work to untangle the dynamics of gene regulation associated with midge physiology.

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. N/A.

The role of microRNAs in responses to drought and heat stress in peanut (Arachis hypogaea).

MicroRNAs (miRNAs) are 21-24nt small RNAs (sRNAs) that negatively regulate protein-coding genes and/or trigger phased small-interfering RNA (phasiRNA) production. Two thousand nine hundred miRNA families, of which ∼40 are deeply conserved, have been identified in ∼80 different plant species genomes. miRNA functions in response to abiotic stresses is less understood than their roles in development. Only seven peanut MIRNA families are documented in miRBase, yet a reference genome assembly is now published and over 480 plant-like MIRNA loci were predicted in the diploid peanut progenitor Arachis duranensis genome. We explored by computational analysis of a leaf sRNA library and publicly available sRNA, degradome, and transcriptome datasets the miRNA and phasiRNA space associated with drought and heat stresses in peanut. We characterized 33 novel candidate and 33 ancient conserved families of MIRNAs and present degradome evidence for their cleavage activities on mRNA targets, including several noncanonical targets and novel phasiRNA-producing noncoding and mRNA loci with validated novel targets such as miR1509 targeting serine/threonine-protein phosphatase7 and miRc20 and ahy-miR3514 targeting penta-tricopeptide repeats (PPRs), in contradistinction to other claims of miR1509/173/7122 superfamily miRNAs indirectly targeting PPRs via TAS-like noncoding RNA loci. We characterized the inverse correlations of significantly differentially expressed drought- and heat-regulated miRNAs, assayed by sRNA blots or transcriptome datasets, with target mRNA expressions in the same datasets. Meta-analysis of an expression atlas and over representation of miRNA target genes in co-expression networks suggest that miRNAs have functions in unique aspects of peanut gynophore development. Genome-wide MIRNA annotation of the published allopolyploid peanut genome can facilitate molecular breeding of value-added traits.

P-054 Significant differences of micro RNA expression pattern in extracellular vesicles between fertile and infertile individuals: new markers of male reproductive status?

Abstract Study question Is the miRNAs cargo of the extracellular vesicles (EVs) present in seminal plasma different between healthy semen donors and infertile patients? Summary answer There are significant differences in the miRNAs expression patterns in the load of seminal EVs between infertile patients and healthy semen donors What is known already Extracellular vesicles (EVs) present in seminal plasma contain a wide range of different proteins, lipids and nucleic acids, such as microRNA. These microRNA can execute their action by regulating gene expression of target cells triggering an important role in different reproductive processes. It is well stablished in different pathologies that the EVs cargo is representative for the functional status of the producer cell. In this sense, the alteration in microRNAs expression patterns in seminal plasma has been associated with spermatogenic impairments, subfertility and azoospermia, indicating that microRNAs in seminal EVs could work as biomarkers of the male fertility status Study design, size, duration This prospective study, performed between March 2021 to December 2022, included 30 semen samples from infertile males seeking infertility treatment (study group), and 7 semen samples from healthy semen donors (control group) from our donor semen bank. All the semen samples from patients showed abnormal seminal parameters (oligo- and/or asthenozoospermia). Men under drug treatment and/or with infectious or chronic diseases were excluded from the study Participants/materials, setting, methods After conventional semen analysis, EVs were isolated from seminal plasma by ultracentrifugation. To confirm EVs isolation, an aliquot of each sample was used for nanoparticle tracking analysis (NTA). Then, microRNAs from EVs were extracted and miRNAseq was performed. Raw data were standardized using counts per millions and ANOVA was performed to assess differences in the miRNAs expression between groups.Target genes were annotated using miRTarBase and functional enrichment analysis was performed by MIENTURNET and GeneCards Main results and the role of chance The presence of EVs after ultracentrifugation of seminal plasma was confirmed by NTA, showing a range size within 40-140 nm. miRNAseq generated reads for 914 annotated miRNAs and significant differences in the expression of 17 of them were found between patient and control samples. These miRNAs target a total of 1310 genes and functional enrichment analysis indicated that they participate in several processes related to cell differentiation, cell proliferation, immunomodulation, cellular adhesion and apoptosis. Specifically, patient group showed significant higher expression of some members of the hsa-let-7 family (p < 0.001 for let-7i-5p, let-7f-5p, let-7c-5p and let-7a-5p; p < 0.05 for let-7b-5p), which are implicated in adaptative immune system activation and differentiation by gene modulation. Results also found significant (p < 0.01) higher expression in patient group of hsa-miR-92a-3p, hsa-miR-200c-3p and hsa-miR-888-5p, which target genes are related to apoptotic processes. Finally, patient group showed significant (p < 0.001) lower expression of hsa-miR-320b and miR-320a-3p, which interact with transcription factor sequences (DLX5, MYC) implicated in cell proliferation and differentiation; and also lower expression in hsa-miR-423-5p (p < 0.01) and miR-423-3p (p < 0.05) that interact with the sequence of transcription factors as p21, a regulator of cell cycle progression at G1, and with RNA-binding proteins involved in ribosome assembly. Limitations, reasons for caution This study included males with dissimilar seminal disorders (astheno, oligo, and teratozoospermia). Therefore, further studies are needed lowering the heterogeneity of the study group. At present, no data about reproductive outcome is available Wider implications of the findings This work suggests that the significant differences found in the expression pattern in microRNAs in seminal EVs between infertile and fertile men may be related with gene modulation in the reproductive processes and could be used as novel biomarkers of male fertility status. Trial registration number 1904-MAD-045-AP

NcOrtho: efficient and reliable identification of miRNA orthologs.

MicroRNAs (miRNAs) are post-transcriptional regulators that finetune gene expression via translational repression or degradation of their target mRNAs. Despite their functional relevance, frameworks for the scalable and accurate detection of miRNA orthologs are missing. Consequently, there is still no comprehensive picture of how miRNAs and their associated regulatory networks have evolved. Here we present ncOrtho, a synteny informed pipeline for the targeted search of miRNA orthologs in unannotated genome sequences. ncOrtho matches miRNA annotations from multi-tissue transcriptomes in precision, while scaling to the analysis of hundreds of custom-selected species. The presence-absence pattern of orthologs to 266 human miRNA families across 402 vertebrate species reveals four bursts of miRNA acquisition, of which the most recent event occurred in the last common ancestor of higher primates. miRNA families are rarely modified or lost, but notable exceptions for both events exist. miRNA co-ortholog numbers faithfully indicate lineage-specific whole genome duplications, and miRNAs are powerful markers for phylogenomic analyses. Their exceptionally low genetic diversity makes them suitable to resolve clades where the phylogenetic signal is blurred by incomplete lineage sorting of ancestral alleles. In summary, ncOrtho allows to routinely consider miRNAs in evolutionary analyses that were thus far reserved to protein-coding genes.

Role of MicroRNAs in Mammalian Reproduction

MicroRNAs are small, endogenous, non-coding RNA molecules that are around 19-25 nucleotides long. Most miRNAs are produced via transcription of DNA sequences into primary miRNAs, precursor miRNAs, and then mature miRNAs. These miRNA molecules base-pair to mRNAs to control gene expression post-transcriptionally. Mammalian genome now has over 2000 annotated miRNAs which are thought to influence one-third of the genes in the genome since each miRNA can regulate hundreds of target genes. miRNAs are involved in morphogenesis, tissue maintenance, cell development, differentiation, apoptosis, and metabolism etc. This review aims to summarize the current understanding of the role of miRNAs in various stages of mammalian reproductive biology and their involvement in reproductive disorders. We have also summarized the recent advances about miRNA and provide an updated overview of the literature, including the most recent and relevant studies.

MiEAA 2023: updates, new functional microRNA sets and improved enrichment visualizations.

MicroRNAs (miRNAs) are small non-coding RNAs that play a critical role in regulating diverse biological processes. Extracting functional insights from a list of miRNAs is challenging, as each miRNA can potentially interact with hundreds of genes. To address this challenge, we developed miEAA, a flexible and comprehensive miRNA enrichment analysis tool based on direct and indirect miRNA annotation. The latest release of miEAA includes a data warehouse of 19 miRNA repositories, covering 10 different organisms and 139 399 functional categories. We have added information on the cellular context of miRNAs, isomiRs, and high-confidence miRNAs to improve the accuracy of the results. We have also improved the representation of aggregated results, including interactive Upset plots to aid users in understanding the interaction among enriched terms or categories. Finally, we demonstrate the functionality of miEAA in the context of ageing and highlight the importance of carefully considering the miRNA input list. MiEAA is free to use and publicly available at https://www.ccb.uni-saarland.de/mieaa/.

Consequences of depleting TNRC6, AGO, and DROSHA proteins on expression of microRNAs.

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.

Abstract 6076: Compartment-specific multiomic characterisation of ovarian carcinosarcoma

Abstract Background: Ovarian carcinosarcoma (OCS) is the most aggressive ovarian cancer type. Risk of relapse and death is high across all patient groups, including those diagnosed at early stage. OCS is biphasic, comprising both carcinomatous and sarcomatous populations, leading to its historic consideration alongside true sarcomas. However, we now recognize that OCS in fact represent metaplastic carcinomas, with the sarcomatous component having undergone complete epithelial to mesenchymal transition (EMT) from carcinoma. Detailed understanding of shared and compartment-specific OCS biology has the potential to identify therapeutic opportunities to improve patient survival. Methods: We recently curated the largest pathologically-confirmed OCS cohort to date. Here, we perform compartment-specific profiling of 12 cases by mRNA sequencing, whole exome sequencing, microRNA (miRNA) profiling and quantification of tumor-infiltrating lymphocytes. These data paint a detailed picture of shared and compartment-specific biology between matched carcinomatous and sarcomatous compartments. Results: The sarcomatous compartments harbored significantly fewer infiltrating CD8+ cells (P=0.006), with a significantly lower CD8+:CD3+ cell ratio compared to the carcinomatous components (P=0.002). In 11 of 12 cases, identical TP53 mutations were identified in both cell populations; the remaining case was TP53 wild-type in both compartments. Paired analysis of mRNA sequencing identified 1477 significantly differentially expressed transcripts at FDR<0.01. The sarcomatous compartments demonstrated significantly higher MAPK activity, as determined by the MPAS transcriptomic score (P=0.042). Principal component analysis and unsupervised hierarchical clustering of miRNA expression grouped samples by compartment (carcinomatous versus sarcomatous), rather than by patient, suggesting global differences in the miRNA landscape. Paired analysis identified 131 significantly differentially expressed miRNAs between the two populations (FDR<0.01). Significantly enriched miRNA gene targets were identified using The MiRNA Enrichment Analysis and Annotation Tool (miEAA), identifying key EMT-associated gene targets, including SIRT1, PRDM16, ZEB1, ZEB2 and TGFB signaling components, among other biological processes. Conclusions: Carcinomatous and sarcomatous compartments of OCS demonstrate marked differences in transcriptomic profiles, immune engagement and miRNA expression patterns. Identification of shared TP53 mutations between compartments supports the notion that OCS represent metaplastic carcinomas. Shared biological events represent opportunities for targeted interventions that may be efficacious against both cell populations, while compartment-specific biological events allude to mechanisms by which the carcinomatous population undergoes EMT to form the sarcomatous compartment. Citation Format: Robert L. Hollis, Ailsa Oswald, Lorna J. Stillie, Ian Croy, Michael Churchman, Charlie Gourley, C. Simon Herrington. Compartment-specific multiomic characterisation of ovarian carcinosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6076.

MicroRNAs of extracellular vesicles derived from mesenchymal stromal cells alleviate inflammation in dry eye disease by targeting the IRAK1/TAB2/NF-κB pathway

PurposeTo investigate the efficacy and mechanisms of human umbilical cord-derived MSC-derived extracellular vesicles (hucMSC-EVs) in a mouse model of desiccation-induced dry eye disease (DED). MethodshucMSC-EVs were enriched by ultracentrifugation. The DED model was induced by desiccating environment combined with scopolamine administration. The DED mice were divided into the hucMSC-EVs group, fluorometholone (FML) group, PBS group, and blank control group. Tear secretion, corneal fluorescein staining, the cytokine profiles in tears and goblet cells, TUNEL-positive cell, and CD4+ cells were examined to assess therapeutic efficiency. The miRNAs in the hucMSC-EVs were sequenced, and the top 10 were used for miRNA enrichment analysis and annotation. The targeted DED-related signaling pathway was further verified by using RT‒qPCR and western blotting. ResultsTreatment with hucMSC-EVs increased the tear volume and maintained corneal integrity in DED mice. The cytokine profile in the tears of the hucMSC-EVs group presented with a lower level of proinflammatory cytokines than PBS group. Moreover, hucMSC-EVs treatment increased goblet cell density and inhibited cell apoptosis and CD4+ cell infiltration. Functional analysis of the top 10 miRNAs in hucMSC-EVs showed a high correlation with immunity. Among them, miR-125 b, let-7b, and miR-6873 were conserved between humans and mice and were associated with the IRAK1/TAB2/NF-κB pathway that was activated in DED. Furthermore, IRAK1/TAB2/NF-κB pathway activation and the abnormal expression of IL-4, IL-8, IL-10, IL-13, IL-17, and TNF-α were reversed by hucMSC-EVs. ConclusionshucMSCs-EVs alleviate DED signs, suppress inflammation and restore homeostasis of the corneal surface by multitargeting the IRAK1/TAB2/NF-κB pathway via certain miRNAs.