tRNA-derived RNA fragment, tRF-18-8R6546D2, promotes pancreatic adenocarcinoma progression by directly targeting ASCL2.

Pancreatic adenocarcinoma (PAAD) is a life-threatening cancer. Exploring new diagnosis and treatment targets helps improve its prognosis. tRNA-derived small non-coding RNAs (tsRNAs) are a novel type of gene expression regulators and their dysregulation is closely related to many human cancers. Yet the expression and functions of tsRNAs in PAAD are not well understood. Our study used RNA sequencing to identify tsRNA expression profiles in PAAD cells cultured in no or high glucose media and found tRF-18-8R6546D2 was an uncharacterized tsRNA, which has significantly high expression in PAAD cells and tissues. Clinically, tRF-18-8R6546D2 is linked to poor prognosis in PAAD patients and can be used to distinguish them from healthy populations. Functionally, in vitro and vivo, tRF-18-8R6546D2 over-expression promoted PAAD cell proliferation, migration and invasion, inhibited apoptosis, whereas tRF-18-8R6546D2 knock-down showed opposite effects. Mechanistically, tRF-18-8R6546D2 promoted PAAD malignancy partly by directly silencing ASCL2 and further regulating its downstream genes such as MYC and CASP3. These findings show that tRF-18-8R6546D2 is a novel oncogenic factor and can be a promising diagnostic or prognostic biomarker and therapeutic target for PAAD.

Non-coding RNA yREX3 from human extracellular vesicles exerts macrophage-mediated cardioprotection via a novel gene-methylating mechanism.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

The Multifaceted Role of miR-21 in Pancreatic Cancers.

With the lack of specific signs and symptoms, pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at late metastatic stages, resulting in poor survival outcomes. Among various biomarkers, microRNA-21 (miR-21), a small non-coding RNA, is highly expressed in PDAC. By inhibiting regulatory proteins at the 3' untranslated regions (UTR), miR-21 holds significant roles in PDAC cell proliferation, epithelial-mesenchymal transition, angiogenesis, as well as cancer invasion, metastasis, and resistance therapy. We conducted a systematic search across major databases for articles on miR-21 and pancreatic cancer mainly published within the last decade, focusing on their diagnostic, prognostic, therapeutic, and biological roles. This rigorous approach ensured a comprehensive review of miR-21's multifaceted role in pancreatic cancers. In this review, we explore the current understandings and future directions regarding the regulation, diagnostic, prognostic, and therapeutic potential of targeting miR-21 in PDAC. This exhaustive review discusses the involvement of miR-21 in proliferation, epithelial-mesenchymal transition (EMT), apoptosis modulation, angiogenesis, and its role in therapy resistance. Also discussed in the review is the interplay between various molecular pathways that contribute to tumor progression, with specific reference to pancreatic ductal adenocarcinoma.

Novel non-invasive molecular signatures for oral cavity cancer, by whole transcriptome and small non-coding RNA sequencing analyses: Predicted association with PI3K/AKT/mTOR pathway.

INTRODUCTION: Identification of molecular biomarkers in the saliva and serum of oral cavity cancer patients represents a first step in the development of essential and efficient clinical tools for early detection and post-treatment monitoring. We hypothesized that molecular analyses of paired saliva and serum samples from an individual would likely yield better results than analyses of either serum or saliva alone. MATERIALS AND METHODS: We performed whole-transcriptome and small non-coding RNA sequencing analyses on 32 samples of saliva and serum collected from the same patients with oral squamous cell carcinoma (OSCC) and healthy controls (HC). RESULTS: We identified 12 novel saliva and serum miRNAs and a panel of unique miRNA and mRNA signatures, significantly differentially expressed in OSCC patients relative to HC (log2 fold change: 2.6-26.8; DE: 0.02-0.000001). We utilized a combined panel of the 10 top-deregulated miRNAs and mRNAs and evaluated their putative diagnostic potential (>87% sensitivity; 100% specificity), recommending seven of them for further validation. We also identified unique saliva and serum miRNAs associated with OSCC and smoking history (OSCC smokers vs. never-smokers or HC: log2 fold change: 22-23; DE: 0.00003-0.000000001). Functional and pathway analyses indicated interactions between the discovered OSCC-related non-invasive miRNAs and mRNAs and their targets, through PI3K/AKT/mTOR signaling. CONCLUSION: Our data support our hypothesis that using paired saliva and serum from the same individuals and deep sequencing analyses can provide unique combined mRNA and miRNA signatures associated with canonical pathways that may have a diagnostic advantage relative to saliva or serum alone and may be useful for clinical testing. We believe this data will contribute to effective preventive care by post-treatment monitoring of patients, as well as suggesting potential targets for therapeutic approaches.

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction.

Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

OBJECTIVE: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). ANIMALS: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. METHODS: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. RESULTS: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. CLINICAL RELEVANCE: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Exploring small non-coding RNAs as blood-based biomarkers to predict Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) diagnosis relies on clinical symptoms complemented with biological biomarkers, the Amyloid Tau Neurodegeneration (ATN) framework. Small non-coding RNA (sncRNA) in the blood have emerged as potential predictors of AD. We identified sncRNA signatures specific to ATN and AD, and evaluated both their contribution to improving AD conversion prediction beyond ATN alone. METHODS: This nested case-control study was conducted within the ACE cohort and included MCI patients matched by sex. Patients free of type 2 diabetes underwent cerebrospinal fluid (CSF) and plasma collection and were followed-up for a median of 2.45-years. Plasma sncRNAs were profiled using small RNA-sequencing. Conditional logistic and Cox regression analyses with elastic net penalties were performed to identify sncRNA signatures for A+(T|N)+ and AD. Weighted scores were computed using cross-validation, and the association of these scores with AD risk was assessed using multivariable Cox regression models. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) enrichment analysis of the identified signatures were performed. RESULTS: The study sample consisted of 192 patients, including 96 A+(T|N)+ and 96 A-T-N- patients. We constructed a classification model based on a 6-miRNAs signature for ATN. The model could classify MCI patients into A-T-N- and A+(T|N)+ groups with an area under the curve of 0.7335 (95% CI, 0.7327 to 0.7342). However, the addition of the model to conventional risk factors did not improve the prediction of AD beyond the conventional model plus ATN status (C-statistic: 0.805 [95% CI, 0.758 to 0.852] compared to 0.829 [95% CI, 0.786, 0.872]). The AD-related 15-sncRNAs signature exhibited better predictive performance than the conventional model plus ATN status (C-statistic: 0.849 [95% CI, 0.808 to 0.890]). When ATN was included in this model, the prediction further improved to 0.875 (95% CI, 0.840 to 0.910). The miRNA-target interaction network and functional analysis, including GO and KEGG pathway enrichment analysis, suggested that the miRNAs in both signatures are involved in neuronal pathways associated with AD. CONCLUSIONS: The AD-related sncRNA signature holds promise in predicting AD conversion, providing insights into early AD development and potential targets for prevention.

Insights into the regulatory role of bacterial sncRNA and its extracellular delivery via OMVs.

Small noncoding RNAs (sncRNAs) play important regulatory roles in bacterial physiological processes and host-pathogen interactions. Meanwhile, bacterial outer membrane vesicles (OMVs), as naturally secreted outer membrane structures, play a vital role in the interaction between bacteria and their living environment, including the host environment. However, most current studies focus on the biological functions of sncRNAs in bacteria or hosts, while neglecting the roles and regulatory mechanisms of the OMVs that encapsulate these sncRNAs. Therefore, this review aims to summarize the intracellular regulatory roles of bacterial sncRNAs in promoting pathogen survival by regulating virulence, modulating bacterial drug resistance, and regulating iron metabolism, and their extracellular regulatory function for influencing host immunity through host-pathogen interactions. Additionally, we introduce the key role played by OMVs, which serve as important cargoes in bacterial sncRNA-host interactions. We propose emerging pathways of sncRNA action to further discuss the mode of host-pathogen interactions, highlighting that the inhibition of sncRNA delivery by OMVs may prevent the occurrence of infection to some extent. Hence, this review lays the foundation for future prophylactic treatments against bacterial infections and strategies for addressing bacterial drug resistance. KEY POINTS: •sncRNAs have intracellular and extracellular regulatory functions in bacterial physiological processes and host-pathogen interactions. •OMVs are potential mediators between bacterial sncRNAs and host cells. •OMVs encapsulating sncRNAs have more potential biological functions.

Integrated small RNA, mRNA and protein omics reveal a miRNA network orchestrating metabolic maturation of the developing human heart.

BACKGROUND: As the fetal heart develops, cardiomyocyte proliferation potential decreases while fatty acid oxidative capacity increases in a highly regulated transition known as cardiac maturation. Small noncoding RNAs, such as microRNAs (miRNAs), contribute to the establishment and control of tissue-specific transcriptional programs. However, small RNA expression dynamics and genome-wide miRNA regulatory networks controlling maturation of the human fetal heart remain poorly understood. RESULTS: Transcriptome profiling of small RNAs revealed the temporal expression patterns of miRNA, piRNA, circRNA, snoRNA, snRNA and tRNA in the developing human heart between 8 and 19 weeks of gestation. Our analysis demonstrated that miRNAs were the most dynamically expressed small RNA species throughout mid-gestation. Cross-referencing differentially expressed miRNAs and mRNAs predicted 6200 mRNA targets, 2134 of which were upregulated and 4066 downregulated as gestation progressed. Moreover, we found that downregulated targets of upregulated miRNAs, including hsa-let-7b, miR-1-3p, miR-133a-3p, miR-143-3p, miR-499a-5p, and miR-30a-5p predominantly control cell cycle progression. In contrast, upregulated targets of downregulated miRNAs, including hsa-miR-1276, miR-183-5p, miR-1229-3p, miR-615-3p, miR-421, miR-200b-3p and miR-18a-3p, are linked to energy sensing and oxidative metabolism. Furthermore, integrating miRNA and mRNA profiles with proteomes and reporter metabolites revealed that proteins encoded in mRNA targets and their associated metabolites mediate fatty acid oxidation and are enriched as the heart develops. CONCLUSIONS: This study presents the first comprehensive analysis of the small RNAome of the maturing human fetal heart. Our findings suggest that coordinated activation and repression of miRNA expression throughout mid-gestation is essential to establish a dynamic miRNA-mRNA-protein network that decreases cardiomyocyte proliferation potential while increasing the oxidative capacity of the maturing human fetal heart. Our results provide novel insights into the molecular control of metabolic maturation of the human fetal heart.

Extracellular vesicle small RNA cargo discriminates non-cancer donors from pediatric B-lymphoblastic leukemia patients.

Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates.

Non-coding RNAs from seminal plasma extracellular vesicles and success of live birth among couples undergoing fertility treatment.

Background: Infertility remains a global health problem with male-factor infertility accounting for around 50% of cases. Understanding the molecular markers for the male contribution of live birth success has been limited. Here, we evaluated the expression levels of seminal plasma extracellular vesicle (spEV) non-coding RNAs (ncRNAs) in men of couples in relation with those with and without a successful live birth after infertility treatment. Method: Sperm-free spEV small RNA profiles were generated from 91 semen samples collected from male participants of couples undergoing assisted reproductive technology (ART) treatment. Couples were classified into two groups based on successful live birth (yes, n = 28) and (no, n = 63). Mapping of reads to human transcriptomes followed the order: miRNA > tRNA > piRNA > rRNA> "other" RNA > circRNA > lncRNA. Differential expression analysis of biotype-specific normalized read counts between groups were assessed using EdgeR (FDR<0.05). Result: We found a total of 12 differentially expressed spEV ncRNAs which included 10 circRNAs and two piRNAs between the live birth groups. Most (n = 8) of the identified circRNAs were downregulated in the no live birth group and targeted genes related to ontology terms such as negative reproductive system and head development, tissue morphogenesis, embryo development ending in birth or egg hatching, and vesicle-mediated transport. The differentially upregulated piRNAs overlapped with genomic regions including coding PID1 genes previously known to play a role in mitochondrion morphogenesis, signal transduction and cellular proliferation. Conclusion: This study identified novel ncRNAs profiles of spEVs differentiating men of couples with and without live birth and emphasizes the role of the male partner for ART success.

Non-Coding RNAs in Human Health and Diseases.

Non-coding RNAs (ncRNAs) are, arguably, the enigma of the RNA transcriptome. Even though there are more annotated ncRNAs (25,967) compared to mRNAs (19,827), we know far less about each of the genes that produce ncRNA, especially in terms of their regulation, molecular functions, and interactions. Further, we are only beginning to understand the role of differential regulation or function of ncRNAs caused by genetic and epigenetic perturbations, such as single nucleotide variants (SNV), deletions, insertions, and histone/DNA modifications. The 22 papers in this Special Issue describe the emerging roles of ncRNAs in neurological, cardiovascular, immune, and hepatic systems, to name a few, as well as in diseases such as cancer, Prader-Willi Syndrome, cardiac arrhythmias, and diabetes. As we begin to understand the function and regulation of this class of RNAs, strategies targeting ncRNAs could lead to improved therapeutic interventions for some conditions.

cDNA Cloning of Feline PIWIL1 and Evaluation of Expression in the Testis of the Domestic Cat.

The PIWI clade of Argonaute proteins is essential for spermatogenesis in all species examined to date. This protein family binds specific classes of small non-coding RNAs known as PIWI-interacting RNAs (piRNAs) which together form piRNA-induced silencing complexes (piRISCs) that are recruited to specific RNA targets through sequence complementarity. These complexes facilitate gene silencing through endonuclease activity and guided recruitment of epigenetic silencing factors. PIWI proteins and piRNAs have been found to play multiple roles in the testis including the maintenance of genomic integrity through transposon silencing and facilitating the turnover of coding RNAs during spermatogenesis. In the present study, we report the first characterization of PIWIL1 in the male domestic cat, a mammalian system predicted to express four PIWI family members. Multiple transcript variants of PIWIL1 were cloned from feline testes cDNA. One isoform shows high homology to PIWIL1 from other mammals, however, the other has characteristics of a "slicer null" isoform, lacking the domain required for endonuclease activity. Expression of PIWIL1 in the male cat appears limited to the testis and correlates with sexual maturity. RNA-immunoprecipitation revealed that feline PIWIL1 binds small RNAs with an average size of 29 nt. Together, these data suggest that the domestic cat has two PIWIL1 isoforms expressed in the mature testis, at least one of which interacts with piRNAs.

MicroRNA-675-5p Overexpression Is an Independent Prognostic Molecular Biomarker of Short-Term Relapse and Poor Overall Survival in Colorectal Cancer.

Colorectal cancer (CRC) is the main cause of cancer-related deaths globally, highlighting the importance of accurate biomarkers for early detection and accurate prognosis. MicroRNAs (miRNAs) have emerged as effective cancer biomarkers. The aim of this study was to investigate the prognostic potential of miR-675-5p as a molecular prognostic biomarker in CRC. For this reason, a quantitative PCR assay was developed and applied to determine miR-675-5p expression in cDNAs from 218 primary CRC and 90 paired normal colorectal tissue samples. To assess the significance of miR-675-5p expression and its association with patient outcome, extensive biostatistical analysis was performed. miR-675-5p expression was found to be significantly downregulated in CRC tissue samples compared to that in adjacent normal colorectal tissues. Moreover, high miR-675-5p expression was associated with shorter disease-free (DFS) and overall survival (OS) in CRC patients, while it maintained its unfavorable prognostic value independently of other established prognostic factors. Furthermore, TNM stage stratification demonstrated that higher miR-675-5p levels were associated with shorter DFS and OS intervals, particularly in patients with CRC of TNM stage II or III. In conclusion, our findings suggest that miR-675-5p overexpression constitutes a promising molecular biomarker of unfavorable prognosis in CRC, independent of other established prognostic factors, including TNM staging.

Small RNA MTS1338 Configures a Stress Resistance Signature in Mycobacterium tuberculosis.

In the course of evolution, Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs (sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host-pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin-antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 "core regulon", consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.

Exosomal hsa-miR-335-5p and hsa-miR-483-5p are novel biomarkers for rheumatoid arthritis: A development and validation study.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes cartilage and bone damage. Exosomes are small extracellular vesicles that play a critical role in intercellular communication and various biological processes by serving as vehicles for the transfer of diverse molecules, such as nucleic acids, proteins, and lipids, between cells. The purpose of this study was to develop potential biomarkers for RA in peripheral blood by performing small non-coding RNA (sncRNA) sequencing using circulating exosomes from healthy controls and patients with RA. METHODS: In this study, we investigated extracellular sncRNAs associated with RA in peripheral blood. Using RNA sequencing and differentially expressed sncRNA analysis, we identified a miRNA signature and target genes. Target gene expression was validated via the four GEO datasets. RESULTS: Exosomal RNAs were successfully isolated from the peripheral blood of 13 patients with RA and 10 healthy controls. The hsa-miR-335-5p and hsa-miR-486-5p expression levels were higher in patients with RA than in controls. We identified the SRSF4 gene, which is a common target of hsa-miR-335-5p and hsa-miR-483-5p. As expected, the expression of this gene was found to be decreased in the synovial tissues of patients with RA through external validation. In addition, hsa-miR-335-5p was positively correlated with antiCCP, DAS28ESR, DAS28CRP, and rheumatoid factor. CONCLUSIONS: Our results provide strong evidence that circulating exosomal miRNA (hsa-miR-335-5p and hsa-miR-486-5p) and SRSF4 could be valuable biomarkers for RA.

Urinary phthalate metabolites and small non-coding RNAs from seminal plasma extracellular vesicles among men undergoing infertility treatment.

Non-coding RNA (ncRNA) cargo of extracellular vesicles (EVs) in the male reproductive tract play critical roles in semen quality and emerging evidence suggests their susceptibility to environmental factors. Male phthalate exposures have been linked to poor semen quality, sperm DNA methylation profiles and embryo development; however, there is limited evidence on their potential impact on EV ncRNAs profiles. We evaluated the association between urinary phthalate metabolites and small ncRNAs (sncRNAs) of seminal plasma EVs (spEV) among men receiving clinical infertility care. We conducted sncRNA sequencing of EVs in 96 seminal plasma samples collected from the Sperm Environmental Epigenetics and Development Study (SEEDS). Sequencing reads were mapped to human transcriptome databases using STAR. Urinary metabolite concentrations of thirteen phthalates and two DiNCH, a phthalate alternative, were measured via tandem mass spectrometry. Associations with normalized counts were assessed using EdgeR (FDR<0.05) adjusting for urinary dilution via specific gravity, age, BMI, batch, and biotype-specific total counts. Select metabolites, MEOHP, MECPP, ∑DEHP, MCPP, MCNP, MCOP, were negatively (p < 0.05) correlated with miRNA relative abundance. Similarly, nine metabolites including MEOHP, MECPP, MEHP, MCPP, MHBP, MHiNCH, MiBP, MEHHP, MCOP and ∑DEHP were associated (q < 0.05) with normalized counts from 23 unique ncRNA transcripts (7 miRNAs (pre & mature); 6 tRFs; and 10 piRNAs), most (78%) of which displayed increased expression patterns. miRNA and tRFs gene targets were enriched in vesicle-mediated transport and developmental-related ontology terms, such as tyrosine kinase, head development, and cell morphogenesis. Six genes (MAPK1, BMPR1A/2, PTEN, TGFBR2, TP53 and APP) were present in all the ontology terms and predicted to form protein association networks. piRNAs were annotated to pseudogenes of genes important in EV cargo transfer and embryonic development. This is the first study to associate phthalate exposures to altered spEV sncRNA profiles. Future studies are needed to determine their impact on reproductive outcomes.

sRNA21, a novel small RNA, protects Mycobacterium abscessus against oxidative stress.

BACKGROUND: During infection, Mycobacterium abscessus encounters numerous environmental changes and adapts to them using a variety of complex mechanisms. Non-coding small RNAs (sRNAs) have been shown in other bacteria to be involved in post-transcriptional regulatory pathways, including environmental stress adaptation. However, the potential role of sRNAs in the resistance to oxidative stress in M. abscessus was not clearly described. METHODS: In the present study, we analyzed putative sRNAs identified by RNA-sequencing (RNA-seq) experiments in M. abscessus ATCC_19977 under oxidative stress, and the transcription profiles of sRNAs with differential expression were verified by quantitative reverse transcription-PCR (qRT-PCR). Six sRNA overexpression strains were constructed, and the differences in growth curves between these strains and the control strain were verified. An upregulated sRNA under oxidative stress was selected and named sRNA21. The survival ability of the sRNA21 overexpression strain was assessed, and computer-based approaches were used to predict the targets and pathways regulated by sRNA21. The total ATP production and NAD+ /NADH ratio of the sRNA21 overexpression strain were measured. The expression level of antioxidase-related genes and the activity of antioxidase were tested to confirm the interaction of sRNA21 with the predicted target genes in silico. RESULTS: In total, 14 putative sRNAs were identified under oxidative stress, and the qRT-PCR analysis of six sRNAs showed comparable results to RNA-seq assays. Overexpression of sRNA21 in M. abscessus increased cell growth rate and intracellular ATP level before and after peroxide exposure. The expression of genes encoding alkyl hydroperoxidase and superoxide dismutase was significantly increased, and superoxide dismutase activity was enhanced in the sRNA21 overexpression strain. Meanwhile, after sRNA21 overexpression, the intracellular NAD+ /NADH ratio decreased, indicating changes in redox homeostasis. CONCLUSIONS: Our findings show that sRNA21 is an oxidative stress-induced sRNA that increases M. abscessus survival and promotes the expression of antioxidant enzymes under oxidative stress. These findings may provide new insights into the adaptive transcriptional response of M. abscessus to oxidative stress.

Ox-LDL induced profound changes of small non-coding RNA in rat endothelial cells.

Introduction: Atherosclerosis (AS) is a common cardiovascular disease with a high incidence rate and mortality. Endothelial cell injury and dysfunction are early markers of AS. Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of AS. Ox-LDL promotes endothelial cell apoptosis and induces inflammation and oxidative stress in endothelial cells. Small non-coding RNAs (sncRNAs) mainly include Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), microRNAs (miRNAs) and repeat-associated RNAs. Studies have shown that small non-coding RNAs play an increasingly important role in diseases. Methods: We used ox-LDL to treat rat endothelial cells to simulate endothelial cell injury. The expression changes of sncRNA were analyzed by small RNA high-throughput sequencing, and the expression changes of piRNA, snoRNA, snRNA, miRNA and repeat-associated RNA were verified by quantitative polymerase chain reaction (qPCR). Results: Small RNA sequencing showed that 42 piRNAs were upregulated and 38 piRNAs were downregulated in endothelial cells treated with ox-LDL. PiRNA DQ614630 promoted the apoptosis of endothelial cells. The snoRNA analysis results showed that 80 snoRNAs were upregulated and 68 snoRNAs were downregulated in endothelial cells with ox-LDL treatment, and snoRNA ENSRNOT00000079032.1 inhibited the apoptosis of endothelial cells. For snRNA, we found that 20 snRNAs were upregulated and 26 snRNAs were downregulated in endothelial cells with ox-LDL treatment, and snRNA ENSRNOT00000081005.1 increased the apoptosis of endothelial cells. Analysis of miRNAs indicated that 106 miRNAs were upregulated and 91 miRNAs were downregulated in endothelial cells with ox-LDL treatment, and miRNA rno-novel-136-mature promoted the apoptosis of endothelial cells. The repeat RNA analysis results showed that 4 repeat RNAs were upregulated and 6 repeat RNAs were downregulated in endothelial cells treated with ox-LDL. Discussion: This study first reported the expression changes of sncRNAs in endothelial cells with ox-LDL treatment, which provided new markers for the diagnosis and treatment of endothelial cell injury.