Myostatin (MSTN) protein, lncRNAs, and circRNAs regulate skeletal muscle growth and development. This work aims to compare the expression patterns of circRNAs and lncRNAs in the gluteus maximus tissue of wild-type (WT) and MSTN gene knockout (KO) rabbits. Within the gluteus maximus tissue of three WT and four MSTN KO rabbits, we analyzed the expression profiles of circRNAs and lncRNAs. After identifying the differently expressed RNAs, the biological pathways implicated were ascertained by performing enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). We identified differences in the expression of 251 circRNAs (79 upregulated and 172 downregulated), 176 lncRNAs (53 upregulated and 123 downregulated), and 1178 mRNAs (408 upregulated and 770 downregulated) between WT and MSTN KO rabbits. Target genes were significantly enriched in pathways associated with protein synthesis and catabolism, such as oxidative phosphorylation, ubiquitin-mediated proteolysis, the FoxO signaling pathway, and the pentose phosphate pathway, as identified through GO and KEGG enrichment analyses. The constructed network indicates that a class of circRNAs and lncRNAs is engaged in MSTN-mediated regulation of skeletal muscle development. These findings provide valuable insights for innovative therapeutic, diagnostic, and preventive approaches to muscle disorders.

Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, represents a unique vulnerability in cancer cells. However, current ferroptosis-inducing therapies face clinical limitations due to poor cancer cell specificity, systemic toxicity, and off-target effects. Therefore, a deeper understanding of molecular regulators of ferroptosis sensitivity is critical for developing targeted therapies. The metabolic plasticity of cancer cells determines their sensitivity to ferroptosis. While mitochondrial dysfunction contributes to metabolic reprogramming in cancer, its role in modulating ferroptosis remains poorly characterized. Previously, studies have identified that mitochondrial genome also encodes several non-coding RNAs. We identified 13 novel mitochondrial genome-encoded miRNAs (mitomiRs) that are aberrantly overexpressed in triple-negative breast cancer (TNBC) cell lines and patient tumors. We observed higher levels of mitomiRs in basal-like triple-negative breast cancer (TNBC) cells compared to mesenchymal stem-like TNBC cells. Strikingly, 11 of these mitomiRs directly target the 3'UTR of ZEB1, a master regulator of epithelial-to-mesenchymal transition (EMT). Using mitomiR-3 mimic, inhibitor and sponges, we demonstrated its role as a key regulator of ZEB1 expression in TNBC cells. Inhibition of mitomiR-3 via sponge construct in basal-like TNBC, MDA-MB-468 cells, promoted ZEB1 upregulation and induced a mesenchymal phenotype. Further, mitomiR-3 inhibition in TNBC cells contributed to reduced cancer cell proliferation, migration, and invasion. Mechanistically, mitomiR-3 inhibition in TNBC cells promote metabolic reprogramming toward pro-ferroptotic pathways, including iron accumulation, increased polyunsaturated fatty acid (PUFA) metabolites, and lipid peroxidation, contributing to ferroptotic cell death via ZEB1-mediated downregulation of GPX4, a critical ferroptosis defense enzyme. We observed that mitomiR-3 inhibition significantly suppressed tumor growth in vivo. Our identified mitomiR-3 has low expression in normal breast cells, minimizing potential off-target toxicity, making them a promising target for pro-ferroptotic cancer therapy. Our study reveals a novel link between mitochondrial miRNAs and ferroptosis sensitivity in TNBC paving a way for miRNA-based therapeutics.

Superficial surgical site infection (SSI) is a significant risk factor for the development of periprosthetic joint infection (PJI), particularly in diabetic patients. A high-glucose microenvironment is observed to compromise phagocytosis by inducing cellular senescence, which leads to impaired antibacterial immune function. Exosomes derived from umbilical cord stem cells (H-Exos) can reverse the immunosuppressive microenvironment by rejuvenating senescent cells, thereby terminating excessive, persistent, and ineffective inflammatory responses. Thus, a novel exosome-based immunotherapeutic antibacterial strategy to reverse fate is proposed. Vancomycin & lysostaphin-loaded exosomes are incorporated in a customizable microneedle patch (ExoV-ExoL@MN) for controlled release, enabling tailored treatments for diverse clinical scenarios. While rejuvenating macrophage senescent phenotype, the antibiotics encapsulated within exosomes can be responsively released by the hemolysin secreted by bacteria, triggering rapid bacterial killing. Post-infection clearance, they induce a shift from M1 to M2 macrophage polarization, thereby enhancing anti-inflammatory and reparative responses. Furthermore, the components can be mixed on demand and at any time, allowing for real-time customization and fabrication directly at the clinic (fabrication@clinic). This strategy reverses the immunosuppressive microenvironment by rejuvenating senescent macrophages and effectively combats bacterial invasion into deep tissues through bacteria-responsive antibiotic release, providing a promising approach for preventing and treating SSI-induced PJI.

Long noncoding RNAs (lncRNAs) can regulate gene expression by "sponging" microRNAs (miRNAs), reducing their inhibitory effects on mRNAs. However, this mechanism has been minimally investigated in preimplantation embryo development. In this study, we revisited existing RNA sequencing and small RNA sequencing data to investigate the role of lncRNAs in in vitro-produced bovine preimplantation embryos. Our findings revealed that although lncRNAs exhibit expression patterns similar to mRNAs, maternal lncRNAs degrade earlier than mRNAs during embryonic genome activation (EGA). Weighted gene co-expression network analysis identified 27 modules of mRNA and lncRNA, with enrichment analysis showing a significant negative correlation between the polycomb repressive complex pathway and blastocyst formation (R2 = -0.98). Additionally, bioinformatics analysis was used to predict and construct lncRNA-miRNA-mRNA networks, highlighting that lncRNAs bind more to miRNAs compared with mRNAs. Moreover, lncRNA-induced lncRNA-miRNA-mRNA axes participated in mRNA degradation and biogenesis around the EGA stage. These interactions became stronger after EGA, especially after the 16-cell stage. Overall, our study provides new insights into lncRNA-mediated regulatory networks during bovine preimplantation development.

Phosgene is still widely used in industrial production nowadays. However, as a toxic gas, accidental exposure to phosgene can lead to acute lung injury (ALI). Our team previously identified a cell subpopulation as CD34+CD45+ cells in the bronchoalveolar lavage fluid (BALF) of rats. CD34+CD45+ cells were demonstrated to possess stem cell properties and alleviate pulmonary inflammation during phosgene-induced acute lung injury (P-ALI). However, how CD34+CD45+ cells contribute to the anti-inflammatory process remains unexplored. Rats with P-ALI were intratracheally administered with CD34+CD45+ cells, and it was found that both the infiltration of macrophages and apoptotic cells were reduced in the lung tissues. The macrophages were polarized to an anti-inflammatory CD45+CD3-CD163+MHC-IIlo phenotype and restored efferocytosis efficiency, with a decreased level of inflammatory cytokines in the BALF. Moreover, it was observed that CD34+CD45+ cells promoted macrophage efferocytosis ex vivo and in vitro. Exosomes derived from CD34+CD45+ cells were further demonstrated to mediate the enhancement of macrophage efferocytosis. The small RNA sequencing analysis suggested that exosomal rno-miR-149-5p contributed to the effect. The transfection of rno-miR-149-5p mimic induced the enhancement of efferocytosis in macrophages as the exosomes did, while rno-miR-149-5p inhibitor attenuated the effect by exosomes. Our findings provide convincing evidence that CD34+CD45+ cells can alleviate ALI by enhancing macrophage efferocytosis, offering valuable insights into their therapeutic potential in managing chemical-induced acute lung injuries.

This study aimed to investigate the antiosteoporotic effects and regulatory mechanisms of estradiol (E2) and vitamin D. MC3T3-E1 cells were treated with E2, vitamin D, or their combination, followed by a systematic assessment of cell proliferation and osteogenic differentiation capacity across the treatment groups. Subsequently, miRNA sequencing was performed to analyze differentially expressed miRNAs between the control and E2&vitamin D groups. The target relationship between miR-351-5p and IRS1 was validated, and the effects of the miR-351-5p/IRS1 axis on osteogenesis and mTOR/NFκB signaling pathway were determined after combination treatment. Additionally, an ovariectomized (OVX) osteoporosis mouse model was established to ‌systematically examine‌ the effects of E2, vitamin D, and their combination on osteoporosis and mTOR/NFκB signaling pathway. E2 and vitamin D synergistically promoted MC3T3-E1 cell proliferation and osteogenic differentiation. miR-351-5p was identified through miRNA sequencing analysis. miR-351-5p was downregulated in MC3T3-E1 cells after E2 and vitamin D combination treatment, and its overexpression partially reversed the effect of the combination treatment on osteogenesis. IRS1 was a target of miR-351-5p. When overexpressed, IRS1 partially mitigated the impact of miR-351-5p overexpression on osteogenesis and mTOR/NFκB signaling pathway under the combination treatment. Furthermore, in vivo experiments demonstrated that E2 and vitamin D could synergistically prevent osteoporosis in OVX mice by inhibiting the mTOR/NFκB signaling pathway. In conclusion, E2 and vitamin D exhibited a synergistic effect in preventing osteoporosis through the miR-351-5p/IRS1 axis and mTOR/NFκB signaling pathway. E2 and vitamin D combination treatment could be a potential anti-osteoporotic strategy for osteoporosis treatment.

Current experimental data on RNA interactions remain limited, particularly for non-coding RNAs, many of which have only recently been discovered and operate within complex regulatory networks. Researchers often rely on in-silico interaction detection algorithms, such as TargetScan, which are based on biochemical sequence alignment. However, these algorithms have limited performance. RNA-seq expression data can provide valuable insights into regulatory networks, especially for understudied interactions such as circRNA-miRNA-mRNA. By integrating RNA-seq data with prior interaction networks obtained experimentally or through in-silico predictions, researchers can discover novel interactions, validate existing ones, and improve interaction prediction accuracy.

This paper introduces Pi-GMIFS, an extension of the generalized monotone incremental forward stagewise (GMIFS) regression algorithm that incorporates prior knowledge. The algorithm first estimates prior response values through a prior-only regression, interpolates between these prior values and the original data, and then applies the GMIFS method. Our experimental results on circRNA-miRNA-mRNA regulatory interaction networks demonstrate that Pi-GMIFS consistently enhances precision and recall in RNA interaction prediction by leveraging implicit information from bulk RNA-seq expression data, outperforming the initial prior knowledge.

Pi-GMIFS is a robust algorithm for inferring acyclic interaction networks when the variable ordering is known. Its effectiveness was confirmed through extensive experimental validation. We proved that RNA-seq data of a representative size help infer previously unknown interactions available in TarBase v9 and improve the quality of circRNA disease annotation.

Endogenous microRNAs have been reported to play critical roles in modulating immune response by regulating RNA silencing through cleavage or translational repression of target mRNAs. This study investigates the involvement of Cgi-miR-96P2m identified from oysters Crassostrea gigas in regulating haemocyte proliferation under Vibrio splendidus stimulation. Cgi-miR-96P2m shares a similar sequence with vertebrate miR-96 family members, while maintains an identical seed region similar to mollusk ones, highlighting its evolutionary conservation. The expression levels of Cgi-miR-96P2m in haemocytes significantly downregulated at 48 h after V. splendidus stimulation. The percentage of EdU + cells in haemocytes was significantly higher in the Cgi-miR-96P2m inhibitor + Vs group compared to the inhibitor NC + Vs group, while significantly lower in the Cgi-miR-96P2m mimics + Vs group compared to the mimics NC + Vs group. Binding sites of Cgi-miR-96P2m were predicted at 2383-2401 bp in the coding sequence region of CgVEGFR, and its binding activity with CgVEGFR mRNA was further proved by the dual-luciferase reporter assay. KEGG enrichment analysis revealed that target genes of Cgi-miR-96P2m are enriched in the MAPK signaling pathway and apoptosis process. Inhibition of Cgi-miR-96P2m resulted in upregulated expression of CgP38, cell cycle-related genes (CgCDK2, CgCDC45) and transcription factors (CgGATA, CgRunx), as well as apoptosis-associated genes such as CgBCL-2. These findings suggest that the Cgi-miR-96P2m negatively regulates haemocyte proliferation by targeting CgVEGFR and influencing the expression of cell cycle and apoptosis-related genes in the immune response of oysters.

MicroRNAs (miRNAs) represent a vital class of small non-coding RNAs that play key regulatory roles in gene expression. Accurate identification of miRNA-mRNA interactions is essential for understanding their biological functions. However, current computational prediction tools suffer from several limitations, including species-specific biases, suboptimal accuracy, high false discovery rates, and incomplete target gene coverage. To address these challenges, we present TarP, a novel miRNA target prediction algorithm employing a Polymorphic structured alignment (PMS) approach. Our method mimics the natural binding process between miRNAs and their target mRNAs by integrating key biological interaction features. The algorithm utilizes five distinct nucleotide-binding motifs to perform a structured decomposition and alignment of potential mRNA targets. Predictions are then rigorously evaluated through a dual scoring system: a Structure (St) coefficient assessing binding conformation and an Energy (En) coefficient evaluating thermodynamic stability, ensuring high-confidence target selection. Using experimentally validated human miRNA-mRNA interaction datasets, we benchmarked TarP against four widely used prediction tools (miRanda, RNAhybrid, PITA, and TargetScan). Comparative analyses demonstrate that TarP achieves superior performance in both sensitivity and specificity, exhibiting enhanced accuracy in positive target identification and improved discrimination between true and false interactions. The TarP algorithm is freely available at: https://github.com/Whimonk/TarP.

MicroRNAs (miRNAs) are crucial regulators of gene expression, which contribute to immune response regulation in various organisms, including crustaceans. To investigate the immunoregulatory roles of miRNAs in Portunus trituberculatus, a comparative miRNAomic analysis of Vibrio parahaemolyticus infection was carried out. Through comparative miRNAomic analysis, we identified 17 differentially expressed miRNAs (DE-miRNAs), of which 12 were upregulated. Subsequently, miRNA-mRNA regulatory network analysis revealed that the DE-miRNAs were enriched in immune-related signaling pathways. Within the miRNA-mRNA regulatory network, miRNA novel0045 was identified as a crucial regulator of the tumor necrosis factor (TNF) pathway via targeting the TNF receptor-associated factor 6 gene. This result was corroborated by our RNA interference assay, confirming the significance of miRNA novel0045 in resistance to V. parahaemolyticus infection. Moreover, miRNA novel0294 was noted to possess cross-kingdom regulatory potential, translocating into bacterial cells and directly inhibiting V. parahaemolyticus proliferation. We validated this finding through fluorescence labeling and confocal microscopy, confirming effective internalization and presence of miRNA within bacterial. These results expand the current understanding of miRNA-mediated immune responses in crustaceans, highlighting the roles of miRNAs in host immune defense and cross-kingdom regulatory function in bacterial infection suppression, and have potential implications in the development of RNA-based antimicrobial strategies.

We first measured the content of chlorfenapyr and tralopyril in silkworm larvae using HPLC, revealing that chlorfenapyr can be biotransformed into tralopyril in silkworms. Then, a differential transcriptomic database of small RNA was constructed through Illumina RNA-Sequencing. qRT-PCR was conducted to determine the expression levels of Bmo-miR-6497-5p and the target CYP450 gene, and Bmo-miR-6497-5p was significantly upregulated in the L3 silkworm larvae 24, 48, and 72 h after they were treated with chlorfenapyr. Furthermore, the target P450 gene CYP337A2 was downregulated at these time points. Dual-luciferase validation revealed that the luciferase activity significantly decreased after Bmo-miR-6497-5p bound to CYP337A2. In addition, miRNA mimics/inhibitor injection and bioassays of chlorfenapyr and tralopyril revealed that the mortality of third silkworm larvae injected with the antagomir of Bmo-miR-6497-5p was significantly increased after exposure to a sublethal concentration of chlorfenapyr. These results imply that Bmo-miR-6497-5p targets CYP337A2, regulating its expression. Also, silkworms increase their tolerance to chlorfenapyr by upregulating Bmo-miR-6497-5p expression, thereby inhibiting the biotransformation of chlorfenapyr to toxic tralopyril catalyzed by CYP337A2. The present study reveals the function of microRNA in silkworm tolerance to chlorfenapyr and improves understanding regarding insecticide resistance in Lepidopteran insects.

Circular RNAs (circRNAs) are a class of non-coding RNAs generated through back splicing. High expression of circRNAs is often associated with numerous abnormal cellular biological processes. However, the regulatory factors of circRNAs are not fully understood.

In this study, we identified PTBP1 as a crucial regulator of circRNA biogenesis through a comprehensive analysis of the whole transcriptome profiles across 10 diverse cell lines. Knockdown of PTBP1 led to a significant decrease in circRNA expression, concomitant with a distinct reduction in cell proliferation. To investigate the regulatory mechanism of PTBP1 on circRNA biogenesis, we constructed a minigene reporter based on SPPL3 gene. The results showed that PTBP1 can bind to the flanking introns of circSPPL3, and the mutation of PTBP1 motif impedes the back splicing of circSPPL3. Subsequently, to demonstrate that this observation is not an exception, the comprehensive regulatory effects of PTBP1 on circRNAs were confirmed by miniGFP, reflecting the necessity of the binding site in the flanking introns. Analysis of data from clinical samples showed that both PTBP1 and circRNAs exhibited substantial upregulation in acute myeloid leukemia, further demonstrating a potential role for PTBP1 in promoting circRNA biogenesis under in vivo conditions. Competitive endogenous RNA (ceRNA) network revealed that PTBP1-associated circRNAs participated in biological processes associated with cell proliferation.

In summary, our study is the first to identify the regulatory effect of PTBP1 on circRNA biogenesis and indicates a possible link between PTBP1 and circRNA expression in leukemia.

Barramundi (Lates calcarifer) are emerging as a key species in warm-water aquaculture worldwide; however, disease outbreaks caused by Vibrio spp. are impeding industry expansion. Climate change is expected to exacerbate this issue by intensifying extreme weather events, including unusually cold temperatures, thereby increasing the risk of disease. In this study, we investigated the combined effect of cold stress and V. harveyi infection on the early transcriptome (mRNA) and microRNA responses of juvenile barramundi to enhance our understanding of host-pathogen interactions. High levels of differential gene expression were observed in fish subjected to cold stress (22 °C) post-infection with V. harveyi, with 3231 differentially expressed genes and an extensive pro-inflammatory immune response. In contrast, most differentially expressed microRNAs were associated with fish infected with V. harveyi housed under optimal temperature conditions (30 °C). MicroRNAs play a crucial role in regulating gene expression, typically through downregulation of target mRNAs. The significant upregulation of miRNAs in barramundi kept at 30 °C, and the lack of miRNA upregulation in cold stressed fish, suggests that cold stress impaired the immune-regulatory capacity of affected fish, resulting in a hyper-inflammatory response that may account for the increased mortality observed. This study is the first dual study of the transcriptome and microRNA response of barramundi to V. harveyi infection and expands understanding of the innate immune response in barramundi and the regulatory role of microRNAs in teleost fish.

In addition to being a major source of animal protein, pigs are an important model for studying development and diseases in humans. Over the past two decades, thousands of high-throughput sequencing studies in pigs have been performed using a variety of tissues from different breeds and developmental stages. However, multi-omics databases specifically designed for pig functional genomics research are still limited. Here, we present PIGOME, a user-friendly database of pig multi-omes. PIGOME currently contains seven types of pig omics datasets, including whole-genome sequencing (WGS), RNA sequencing (RNA-seq), microRNA sequencing (miRNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin sequencing (ATAC-seq), bisulfite sequencing (BS-seq), and methylated RNA immunoprecipitation sequencing (MeRIP-seq), from 6901 samples and 392 projects with manually curated metadata, integrated gene annotation, and quantitative trait locus information. Furthermore, various "Explore" and "Browse" functions have been established to provide user-friendly access to omics information. PIGOME implements several tools to visualize genomic variants, gene expression, and epigenetic signals of a given gene in the pig genome, enabling efficient exploration of spatiotemporal gene expression/epigenetic patterns, functions, regulatory mechanisms, and associated economic traits. Collectively, PIGOME provides valuable resources for pig breeding and is helpful for human biomedical research. PIGOME is available at https://pigome.com.

The severe Asiatic form of huanglongbing (HLB), caused by "Candidatus Liberibacter asiaticus" (CLas), threatens global citrus production via the citrus psyllid, Diaphorina citri. Culturing challenges of CLas necessitate reducing D. citri populations for disease management. CLas boosts the fecundity of CLas-positive (CLas+) D. citri and fosters its own proliferation by modulating the insect host's juvenile hormone (JH), but the intricate endocrine regulatory mechanisms remain elusive. Here, it is reported that the D. citri ecdysis-triggering hormone (DcETH) and its receptor DcETHR play pivotal roles in the reciprocal benefits between CLas and D. citri within the ovaries, influencing energy metabolism and reproductive development in host insects; miR-210, negatively regulates DcETHR expression, contributing to this symbiotic interaction. CLas infection reduces 20-hydroxyecdysone (20E) levels and stimulates DcETH release, elevating JH production via DcETHR, enhancing fecundity and CLas proliferation. Furthermore, circulating JH levels suppress 20E production in CLas+ ovaries. Collectively, the orchestrated functional interplay involving 20E, ETH, and JH increases energy metabolism and promotes the fecundity of CLas+ D. citri and CLas proliferation. These insights not only broaden the knowledge of how plant pathogens manipulate the reproductive behavior of insect hosts but also offer novel targets and strategies for combatting HLB and D. citri.

The Japanese flounder (Paralichthys olivaceus) is greatly influenced in terms of muscle quality and quantity by the development of skeletal muscle. While the mechanisms underlying skeletal muscle development are well-studied, the role of non-coding RNAs, particularly circRNAs, in the skeletal muscle development of Japanese flounder remains unclear. To investigate the expression patterns of circRNAs during different developmental stages (JP1: 7 days, JP2: 90 days, JP3: 24 months (female), JP4: 24 months (male)) in Japanese flounder, we performed transcriptome sequencing analysis. We identified a total of 3523 circRNAs, of which 10.19 % were differentially expressed. These differentially expressed (DE) circRNAs were studied, and their impacts on muscle development were analyzed. The RNA interaction network revealed that skeletal muscle-specific circchd6 targeted novel-miR-508 and further regulated dual specificity tyrosine-phosphorylation regulated kinase 2 (dyrk2). Functional analysis showed that overexpressed circchd6 and dyrk2 promoted myoblast proliferation and differentiation, while novel-miR-508 inhibited both. Our study identified the circchd6-novel-miR-508-dyrk2 axis as a regulatory mechanism and provided new evidence for the use of epigenetic approaches in genetic breeding.

MicroRNAs (miRNA) are a class of endogenous non-coding small RNA molecules that are widely found in tissues, biological fluids, and vesicles such as exosomes. Exosomes are extracellular vesicles released from multivesicular bodies of various cell types. They are involved in intercellular communication and transport and immune regulation and may serve as potential biomarkers for diagnosis and monitoring. The function of exosomal miRNAs and their potential applications as biomarkers are a topic of interest. However, identification and comparison of miRNA expression in different biological sample types have rarely been studied. Therefore, in this study, the miRNA profiles of tissue- and tissue-derived exosomes of Pinctada fucata were characterized and compared to screen for differentially expressed miRNAs. The miRNAs functioned within tissues and were also packaged into exosomes. Simultaneously, some miRNAs were preferentially exported to exosomes for their biological functions. Functional analyses suggested that the predicted genes targeted by these differentially expressed miRNAs were extensively involved in intracellular vesicle trafficking and vesicle-mediated substrate transport. Overall, our findings provide insights into the roles of tissue-derived miRNAs and circulating exosomal miRNAs in cell communication and gene regulation. Moreover, this study serves as an additional reference for sample type selection for P. fucata small RNA analysis.

Biomarkers in pleural fluid are the potential auxiliary diagnostic markers for malignant pleural effusion (MPE). Exosomal microRNAs (miRNAs) represent novel diagnostic markers for various diseases. The diagnostic performance of exosomal miRNAs for MPE remains unclear. Therefore, we examined the exosomal miRNAs profiles of both MPE and benign pleural effusion (BPE), aiming to study diagnostic performance of exosomal miRNAs for MPE.

We used next-generation sequencing (NGS) technology to analyze the pleural fluid exosomal miRNA profile in five MPE and 15 BPE cases. We analyzed the differentially expressed exosomal miRNAs by reverse transcription polymerase chain reaction (RT-PCR), with cel-miR-39 or snRNA U6 as internal references. We assessed the diagnostic accuracy of exosomal miRNA for MPE with a receiver operating characteristic (ROC) curve. We also analyzed whether exosomal miRNA could improve the diagnostic performance of pleural carcinoembryonic antigen (CEA).

Fifty-eight miRNAs were up-regulated, and 35 miRNAs were down-regulated in MPE. We selected exosomal miR-182-5p for further study and analyzed miR-182-5p in 153 patients with undiagnosed pleural effusion. Exosomal miR-182-5p was undetectable in 32 participants. In the remaining participants with 49 MPE and 72 BPE cases, we found that the areas under the curve (AUCs) and their 95% confidence intervals (95% CIs) for exosomal miR-182-5p were 0.78 (95% CI: 0.69-0.86) when using cel-miR-39 as an internal reference, and 0.80 (95% CI: 0.73-0.88) when using snRNA U6. The combination of exosomal miR-182-5p and CEA can slightly improve the diagnostic accuracy of MPE, with an AUC of 0.91 (95% CI: 0.85-0.97).

Pleural miR-182-5p can assist in the diagnosis of MPE. Its diagnostic performance is slightly affected by internal reference.

Extracellular vesicle (EV)-mediated small RNA trafficking plays an important role in intercellular and interspecies communication. Plant arboviruses keep homeostasis in insect vectors, thus ensuring vector survival and viral transmission. How plant EV-mediated cross-kingdom RNA interference participates in viral infection in insect vectors remains unknown. Here, we successfully isolate rice EVs and identify a batch of microRNAs (miRNAs) encapsulated in EVs. Two EV-enriched rice miRNAs, Osa-miR159a.1-1 and Osa-miR167a, are transported into midgut epithelial cells of small brown planthopper, which is a competent vector of rice stripe virus (RSV). Osa-miR159a.1-1 elevates the expression of a phospholipase C by enhancing its mRNA stability, inducing the downstream CSL expression to inhibit apoptosis for the benefit of RSV replication. On the other hand, Osa-miR167a directly binds RSV RdRp to suppress viral replication. This differential regulation of EV-mediated cross-kingdom RNA interference contributes to arbovirus homeostasis in insect vectors and the following efficient transmission.

Coral reef ecosystems face escalating threats from anthropogenic global climate challenges, leading to frequent bleaching events. A key issue in coral transplantation is the inability of fragments to rapidly grow to sizes that can resist environmental pressures. The observation of accelerated growth during the early stages of coral regeneration provides new insights for addressing this challenge. To investigate the underlying molecular mechanisms, we study the fast-growing stony coral Acropora muricata. Using single-cell RNA sequencing, bulk RNA sequencing, and high-resolution micro-computed tomography, we identify a critical regeneration phase around 2-4 weeks post-injury. Single-cell transcriptome analysis reveals 11 function-specific cell clusters. Pseudotime analysis indicates epidermal cell differentiation into calicoblasts. Bulk RNA-seq results highlight a temporal limitation in coral's rapid regeneration. Through integrated multi-omics analysis, this study emphasizes the importance of a comprehensive understanding of coral regeneration, providing insights beyond fundamental knowledge and offering potential protective strategies to promote coral growth.

Manila clam plays a crucial role in China's marine aquaculture industry. However, frequent vibriosis outbreaks severely hinder sustainable and healthy development of the shellfish aquaculture industry. This study indicated markedly decreased clam survival rates after 48 h of Vibrio alginolyticus challenge. Gill and hepatopancreas damage was investigated through histological observation. The activity of lysozyme in the gills and hepatopancreas peaked at 12 and 24 h, respectively. V. alginolyticus showed a maximum bacterial load in the gills and hepatopancreas at 12 and 24 h, respectively. Additionally, transcriptome sequencing of hepatopancreas revealed ten differentially expressed miRNAs in Va and Cn after 48 h infection with V. alginolyticus, corresponding to 100 target genes, with eight upregulated and two downregulated DE miRNAs. Gene ontology (GO) enrichment analysis identified 50 known miRNAs and 111 novel miRNAs, thereby predicting a total of 1840 target genes. KEGG analysis revealed significant changes in multiple signaling pathways, involving lysosomes, apoptosis, amino acid metabolism, and endocytosis, in response to V. alginolyticus stimulation. This study provided new information regarding the immune regulation mechanisms of R. philippinarum in response to V. alginolyticus stress.

Muscle fibers, as the fundamental units of muscle tissue, play a crucial role in determining skeletal muscle function through their growth, development, and composition. To investigate changes in muscle fiber types and their regulatory mechanisms in Mongolian horses (MG), Xilingol horses (XL), and Grassland-Thoroughbreds (CY), we conducted histological and bioinformatic analyses on the gluteus medius muscle of these three horse breeds. Immunofluorescence analysis revealed that Grassland-Thoroughbreds had the highest proportion of fast-twitch muscle fibers at 78.63%, while Mongolian horses had the lowest proportion at 57.54%. Whole-transcriptome analysis identified 105 differentially expressed genes (DEGs) in the CY vs. MG comparison and 104 DEGs in the CY vs. XL comparison. Time-series expression profiling grouped the DEGs into eight gene sets, with three sets showing significantly up-regulated or down-regulated expression patterns (p < 0.05). Additionally, 280 differentially expressed long non-coding RNAs (DELs) were identified in CY vs. MG, and 213 DELs were identified in CY vs. XL. A total of 32 differentially expressed microRNAs (DEMIRs) were identified in CY vs. MG, while 44 DEMIRs were found in CY vs. XL. Functional enrichment analysis indicated that the DEGs were significantly enriched in essential biological processes, such as actin filament organization, muscle contraction, and protein phosphorylation. KEGG pathway analysis showed their involvement in key signaling pathways, including the mTOR signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. Furthermore, functional variation-based analyses revealed associations between non-coding RNAs and mRNAs, with some non-coding RNAs targeting genes potentially related to muscle function regulation. These findings provide valuable insights into the molecular basis for the environmental adaptability, athletic performance, and muscle characteristics in horses, offering new perspectives for the breeding of Grassland-Thoroughbreds.

Autism spectrum disorder (ASD) is a brain developmental disability with a not-fully clarified etiogenesis. Current ASD research largely focuses on coding regions of the genome, but up to date much less is known about the contribution of non-coding elements to ASD risk. The non-coding genome is largely made of DNA repetitive sequences (RS). Although RS were considered slightly more than "junk DNA", today RS have a recognized role in almost every aspect of human biology, especially in developing human brain. Our aim was to test if RS transcription may play a role in ASD.

Global RS transcription was firstly investigated in postmortem dorsolateral prefrontal cortex of 13 ASD patients and 39 matched controls. Results were validated in independent datasets.

AmnSINE1 was the only RS significantly downregulated in ASD specimens. The role of AmnSINE1 in ASD has been investigated at multiple levels, showing that the 1,416 genes containing AmnSINE1 are associated with nervous system development and autism susceptibility. This has been confirmed in a different experimental setting, such as in organoid models of the human cerebral cortex, harboring different ASD causative mutations. AmnSINE1 related genes are transcriptionally co-regulated and are involved not only in brain formation but can specifically be involved in ASD development. Looking for a possible direct role of AmnSINE1 non-coding transcripts in ASD, we report that AmnSINE1 transcripts may alter the miRNA regulatory landscape for genes involved in neurogenesis.

Our findings provide preliminary evidence supporting a role for AmnSINE1 in ASD development.

Sugarcane (Saccharum spp.) hybrid, one of the most important crops in Florida, has been affected by orange rust (OR) disease caused by Puccinia kuehnii since 2007, resulting in significant yield loss. Developing resistant cultivars to this disease has become an important goal in sugarcane breeding programs. However, the specific genes and molecular mechanisms underlying the resistance to OR disease in sugarcane are still not clear. In this study, we selected two sugarcane sister lines with different genotypes-showing contrasting resistance responses to the disease-from a major quantitative trait loci (QTL) region controlling OR disease resistance. Morphological and anatomical observations revealed that the resistant line (540) had significantly smaller stomatal size and lower stomatal density than the susceptible line (664). Transcriptomic analyses showed that resistant line 540 had increased cell surface modification activity, suggesting possible increased surface receptors. Differentially expressed gene and coexpression analyses also revealed key genes involved in the biosynthesis of anti-fungal molecules, such as hordatines, arabidopyrones, and alkaloids. They also showed a strong increase in long non-coding RNA expression, playing a role in transcriptional regulation. Transcriptomic-metabolomic joint analysis suggested that the biosynthesis of phenylpropanoid derivatives with purported antioxidant and anti-fungal capabilities increased in line 540, especially those deriving from ferulate. Genes, pathways, and some single-nucleotide polymorphisms identified in this study will provide fundamental information and resources to advance the knowledge of sugarcane molecular genetic mechanisms in relation to OR disease, supporting breeding programs in developing cultivars with improved resistance to OR.

Burn injuries are prevalent, yet effective treatments remain elusive. Exosomes derived from mesenchymal stem cells (MSC-Ex) possess remarkable pro-regenerative properties for wound healing. Despite their potential, the challenge of mass production limits their clinical application. To address this, preparing exosome-like vesicles has become an international trend. In this study, 28 key microRNAs (miRNAs) with significant pro-proliferation, anti-inflammation, and anti-fibrosis functions were screened from MSC-Ex. These miRNAs were encapsulated into liposomes and then hybridized with extracellular vesicles derived from watermelon to create synthetic exosome-like vesicles. The fabricated vesicles exhibited similar particle size and zeta potential to MSC-Ex, demonstrating high serum stability and effectively resisting the degradation of miRNA by RNase. They were efficiently internalized by cells and enabled a high rate of lysosomal escape for miRNAs post cellular uptake, thereby effectively exerting their pro-proliferative, anti-inflammatory, and anti-fibrotic functions. Further experiments demonstrated that these vesicles efficiently accelerated burn wound healing and reduced scarring, with effects comparable to those of natural MSC-Ex. Based on these findings, the exosome-like vesicles fabricated in this study present a promising alternative to MSC-Ex in burn wound treatment.

Circular RNAs (circRNAs) have re-emerged as promising gene regulators in various physiological and pathological conditions. However, the expression patterns of circRNAs in the developing spinal cord of mammals and the comprehensive distribution of circRNAs across different tissues remain poorly understood. In this study, rats were used as the model organism. We conducted a comprehensive analysis of 15 RNA-Seq datasets comprising 217 rat samples and developed a web-based resource, CiRNat, to facilitate access to these data. We identified 15,251 credible circRNAs and validated them through experimental approaches. Notably, we observed two significant time points for circRNA increase during spinal cord development, approximately at embryonic day 14 (E14d) and postnatal week 4 (P4w). Analysis of circRNA expression in various rat tissues revealed higher expression levels in central nervous system tissues compared to peripheral nervous system tissues and other tissues. Furthermore, some highly abundant circRNAs exhibited tissue- and species-specific expression patterns and differed from their cognate linear RNAs, such as those derived from Gigyf2. Integrating polysome profiling and bioinformatic predictions suggested potential functions of certain circRNAs as miRNA sponges and translational templates. Collectively, this study provides the first comprehensive landscape of circRNAs in the developing spinal cord, offering an important resource and new insights for future exploration of functional circRNAs in central nervous system development and related diseases.

The parasitic protozoan Entamoeba histolytica secretes extracellular vesicles (EVs), but so far little is known about their function in the interaction with the host immune system. Infection with E. histolytica trophozoites can lead to formation of amebic liver abscesses (ALAs), in which pro-inflammatory immune responses of Ly6Chi monocytes contribute to liver damage. Men exhibit a more severe pathology as the result of higher monocyte recruitment and a stronger immune response. To investigate the role of EVs and pathogenicity in the host immune response, we studied the effect of EVs secreted by low pathogenic EhA1 and highly pathogenic EhB2 amebae on monocytes. Size and quantity of isolated EVs from both clones were similar. However, they differed in their proteome and miRNA cargo, providing insight into factors potentially involved in amebic pathogenicity. In addition, EVs were enriched in proteins with signaling peptides compared with the total protein content of trophozoites. Exposure to EVs from both clones induced monocyte activation and a pro-inflammatory immune response as evidenced by increased surface presentation of the activation marker CD38 and upregulated gene expression of key signaling pathways (including NF-κB, IL-17 and TNF signaling). The release of pro-inflammatory cytokines was increased in EV-stimulated monocytes and more so in male- than in female-derived cells. While EhA1 EV stimulation caused elevated myeloperoxidase (MPO) release by both monocytes and neutrophils, EhB2 EV stimulation did not, indicating the protective role of MPO during amebiasis. Collectively, our results suggest that parasite-released EVs contribute to the male-biased immunopathology mediated by pro-inflammatory monocytes during ALA formation.

Astyanax mexicanus, a species with both surface-dwelling and multiple cave-dwelling populations, offers a unique opportunity to study repeated adaptation to dark and resource-scarce environments. While previous work has identified large-scale gene expression changes between morphs under even identical laboratory conditions, the regulatory basis of these expression differences remains largely unexplored. In this study, we focus on microRNAs (miRNAs) as key regulators of gene expression. Our analysis identified 683 mature miRNAs, establishing the first comprehensive catalog of miRNAs for this species. We identified a unique subset of differentially expressed miRNAs common to all studied cave-dwelling populations, potentially orchestrating the nuanced gene expression patterns required for survival in the cave milieu. Furthermore, we performed in silico target prediction of these miRNAs, revealing possible roles in developmental and metabolic pathways pivotal for thriving in nutrient-limited cave conditions. Interestingly, we also observed that Molino, which is the "youngest" of the three cavefish analyzed in this study, exhibited the most abundant number of differentially expressed mature miRNAs among the cave morphs. The comprehensive miRNA catalog generated, along with the insight into their differential expression across different morphs, will guide future investigations into the intricate world of miRNA-mediated evolution of complex traits.

Although many sequence variants have been discovered in cattle, deciphering the relationship between genome and phenome remains a significant challenge. In this study, we identified functional classes, including mammary-specific genes, lactation-associated genes, novel long non-coding RNAs, miRNAs, RNA editing sites, DNA methylation, histone modifications, and expression quantitative trait loci. We estimated their contributions to genetic variance for milk production traits using 3 million variants in 23,566 Holstein bulls. Sequence variants in the 5'-UTR, synonymous, and splicing regions disproportionately contributed to genetic variance of milk production traits compared to other genomic regions. Genes specifically expressed in the mammary gland, particularly those active in lactating tissue (e.g., GLYCAM1, DGAT1), account for significantly more genetic variance of milk production traits than specific genes from non-mammary tissues. We identified 8,560 differentially expressed genes (DEGs) between lactating and non-lactating tissues. Among these, both up-regulated and small-fold changes of down-regulated DEGs exhibited greater genetic variance enrichment of milk production traits than other genes. Mammary enhancers (e.g., H3K27ac, H3K4Me1) explained more variance than repressive elements, while small changes in DNA methylation level (≤0.2) contributed more variance than that with larger changes (> 0.2). Notably, lactation-associated RNA editing sites in mammary explained more variance for milk production traits than expected by chance. We proposed a novel miRNA prioritization strategy for selecting candidate miRNAs related to milk production traits, based on the overlaps between significant enrichment tests of miRNA target correlations and the relatively large variance explained by these targets. Additionally, we integrated these nine functional classes into the variance component analysis simultaneously, revealing that sQTLs, histone modification and DEGs showed the highest per-SNP variance enrichment. Finally, we constructed a new 624K SNP panel, which improved the reliabilities of genomic predictions by 0.22%. Dividing routine SNPs into two groups based on functional classes improved the reliabilities by 0.21%, particularly for milk protein percentage (0.68% improvement). Overall, incorporating prior biological knowledge of the mammary gland directly enhances our understanding of milk production's genetic architecture and improves the reliability of genomic predictions for milk production traits. This integrative approach establishes a paradigm for translating biological knowledge into agricultural genomics applications.

20-Hydroxyecdysone (20E) is an important hormone that regulates insect development and metamorphosis. The fat body of insects plays a crucial role in nutrient storage and energy metabolism and is considered the exchange center for regulating insect development. The fat body undergoes remarkable transformation during insect metamorphosis and is primarily regulated by 20E. microRNAs (miRNAs) have been identified in different insects and have multiple functions in various physiological processes. However, the interaction of 20E and miRNAs in fat body regulation remains unclear.

We constructed six small RNA libraries using Bombyx mori fat body treated with 20E. Expression and functional analyses were conducted to identify 20E-responsive miRNAs. In total, 431 miRNAs were identified, including 389 known and 42 novel miRNAs. Differential expression analysis revealed significant expression changes in the expression of 40, 9, and 18 miRNAs at 2 h, 6 h, and 12 h after 20E treatment, respectively. The expression of 10 miRNAs was validated using quantitative real-time PCR. miR-34-5p is a highly conserved miRNA among the 10 validated miRNAs, and autophagy-related gene 1 (Atg1) was considered a target gene of miR-34-5p. The expression analysis of miR-34-5p and Atg1 exhibited an opposite expression pattern in the fat body after the 20E treatment. Dual-luciferase assay indicated that miR-34-5p could inhibit Atg1 expression by targeting a binding site in CDS region of Atg1. In larval fat body, overexpressing miR-34-5p by injecting miR-34-5p agomir suppressed the expression of Atg1 and autophagy, whereas knocking down miR-34-5p by injecting miR-34-5p antagomir induced the expression of Atg1 and autophagy. Meanwhile, Atg1 silencing by RNAi also inhibited autophagy. These results indicate that miR-34-5p participates in 20E-induced autophagy in B. mori fat body by interacting with Atg1.

We systematically identified and functionally characterized miRNAs associated with 20E regulation in the fat body of B. mori. miR-34-5p is involved in 20E-induced autophagy in B. mori by regulating its target gene Atg1. These results provide insight into the role of sophisticated interactions between miRNAs, 20E regulation, and autophagy in fat body remodeling and insect metamorphosis.

Reproduction and breeding are crucial to maintaining abalone aquaculture. Understanding the molecular underpinnings of sexual maturation is essential for advancing knowledge in reproductive biology. However, the molecular mechanisms of gonadal development in abalones remain poorly understood, particularly in microRNA (miRNA)-mediated regulation. Thus, this study conducted a comprehensive transcriptomic analysis of abalone Haliotis discus hannai (H. discus hannai) to identify genes and miRNAs associated with ovarian and testicular discovery. This study identified 685 differentially expressed (DE) genes between the H. discus hannai ovary (DD_ovary) and testis (DD_testis) groups, comprising 479 upregulated and 206 downregulated genes in the DD_ovary. Moreover, 137 miRNAs, including 83 novel and 54 known miRNAs, were detected, with 30 upregulated and 27 downregulated in the DD_ovary compared to the DD_testis. Bioinformatics analysis revealed that these miRNAs regulate key processes such as carbohydrate metabolic processes, kinase and hydrolase activity, and starch and sucrose metabolism, all potentially associated with reproductive traits. Further, key mRNA candidates, including Vitelline envelope sperm lysin receptor (Verl) and Testis-specific serine/threonine-protein kinase (Tssk) 1, and miRNAs such as novel_90 and novel_120, were identified as components of a functional miRNA-mRNA network associated with sexual maturity and sex determination. These key genes were verified using qRT-PCR and fluorescence in situ hybridization (FISH). These transcriptomic and miRNA datasets provide valuable resources for understanding abalone reproductive biology and may support molecular breeding strategies.

The normal development of the testis is essential for male reproduction, as it is the site of sperm production and a prerequisite for spermatogenesis. MiRNAs play crucial roles in various testicular biological processes, including cell proliferation, spermatogenesis, hormone secretion, metabolism, and reproductive regulation. In this study, we utilized deep sequencing data to analyze the expression patterns of small RNAs in testicular tissues of Southern × Hu sheep F1 hybrids at 0, 3, 6 months, and 1 year of age, thereby exploring the functions of miRNAs in testicular development and spermatogenesis. A total of 787 known miRNAs and 415 novel miRNAs were identified. We identified 217, 254, 405, 130, 305, and 138 DE miRNAs in the testes of M0 vs. M3, M0 vs. M6, M0 vs. Y1, M3 vs. M6, M3 vs. Y1, and M6 vs. Y1, respectively. GO annotation and KEGG pathway analysis of DE miRNA target genes revealed that target genes such as YAP1, ITGB1, DOT1L, SMAD4, and SOX9 may be involved in various biological processes, including reproductive pathways such as FOXO, Hippo, Wnt, cAMP, Rap1, and MAPK signaling pathways. The expression levels of 12 randomly selected miRNAs in testes at 0, 3, 6 months, and 1 year of age were detected by qRT-PCR, and the results were consistent with the sequencing data. This study characterized and investigated the differential expression of miRNAs in sheep testes at different developmental stages using deep sequencing technology. These findings will contribute to a deeper understanding of the functions of miRNAs in regulating testicular development and enhancing reproductive performance in male sheep.

Bone healing is a well-orchestrated process involving various bone cells and signaling pathways, where disruptions can result in delayed or incomplete healing. MicroRNAs (miRNAs) are small non-coding RNAs capable of influencing various cellular processes, including bone remodeling. Due to their biological relevance and stable presence in biofluids, miRNAs may serve as candidates for diagnosis and prognosis of delayed bone healing. The aim of the study was to investigate changes in miRNAs circulating in the blood during the healing of rat calvaria defects as biomarkers of successful bone regeneration.

Standardized calvaria defects were created in 36 Wistar rats with a trephine drill and treated with collagen hydroxyapatite (CHA) scaffolds. The treatment groups included CHA scaffolds only, CHA scaffolds containing a plasmid coding for bone morphogenetic protein 2 (BMP2) and miR-590-5p, CHA scaffolds containing mesenchymal stromal cell-derived extracellular vesicles, and empty defects as a control group. After 1, 4 and 8 weeks of healing, the animals were evaluated by microcomputed tomography (microCT), as well as subjected to histological analyses. Blood was sampled from the tail vein prior to surgeries and after 1, 4, and 8 weeks of healing. miRNAs circulating in the plasma were determined using next-generation sequencing.

Variability of bone regeneration within the four groups was unexpectedly high and did not result in significant differences between the groups, as indicated by the microCT and histological analyses of the newly formed bone tissue. However, irrespective of the treatment group and regenerative activity, we identified miRNAs with distinct expression patterns of up- and downregulation at different time points. Furthermore, rats with high and low regenerative activity were characterized by distinct circulating miRNA profiles. miR-133-3p was identified as the top upregulated miRNA and miR-375-3p was identified as the top downregulated miRNA in animals exhibiting strong regeneration over all time points evaluated.

Our study indicates that regardless of the treatment group, success or lack of bone regeneration is associated with a distinct expression pattern of circulating microRNAs. Further research is needed to determine whether their levels in the blood can be used as predictive factors of successful bone regeneration.

Postoperative delirium (POD) is a serious neuropsychiatric complication in elderly surgical patients, yet its pathogenesis remains incompletely understood. Transfer RNA-derived small RNAs (tsRNAs) have emerged as crucial regulators in neurological disorders. We investigated whether specific tsRNAs could serve as predictive biomarkers for POD.

This study conducted a prospective case-control study of 158 elderly patients (≥60 years) undergoing orthopedic surgery. Plasma samples were collected preoperatively and on postoperative day 3.tsRNA expression profiles were analyzed using RNA sequencing and validated by RT-qPCR. Propensity score matching was performed to balance demographic and clinical variables. The predictive value of candidate tsRNAs was assessed using ROC analysis, and their potential functions were explored through bioinformatic analyses.

Among 128 non-POD and 30 POD patients, two tsRNAs (Other-14: 31-tRNA-Gly-CCC-3 and Other-39: 73-tRNA-Arg-TCG-5) showed significantly elevated preoperative levels in POD patients (p<0.001).ROC analysis revealed strong predictive performance (AUC=0.868 and 0.956, respectively).These differences persisted in the propensity-matched cohort (29 pairs).Bioinformatic analyses indicated enrichment in pathways related to neurotransmission, inflammation, and metabolism.

This study identified novel tsRNA biomarkers that robustly predict POD risk and provide insights into its molecular pathogenesis. These findings may facilitate early risk stratification and preventive interventions.