Search Results (18,171)

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators of hepatic metabolism and disease progression. The hepatocyte nuclear factor 1 alpha antisense 1 (HNF1A-AS1) lncRNA modulates liver-specific transcription factors; however, its physiological role in diet-dependent lipid homeostasis remains poorly defined. Methods: In this study, we investigated the mouse ortholog, Hnf1a opposite strand 1 (Hnf1aos1), using AAV-mediated knockdown in C57BL/6J mice fed either a chow diet (10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for 12 weeks. Metabolic phenotyping included hepatic lipid quantification, histological analysis, serum biochemistry, and quantitative gene expression profiling. Results: Loss of Hnf1aos1 produced distinct, diet-dependent alterations in hepatic lipid handling. Under chow conditions, knockdown mice exhibited selective hepatic cholesterol accumulation (6.10 ± 2.9 mg/g tissue vs. 3.51 ± 1.1 mg/g in controls), accompanied by dysregulation of cholesterol clearance pathways. In contrast, under HFD conditions, knockdown precipitated severe macrovesicular degeneration, with hepatic triglyceride levels approximately doubled relative to HFD-fed controls (51.72 ± 19.8 mg/g vs. 26.34 ± 11.9 mg/g) and a numerically elevated triglyceride-to-cholesterol ratio (TG:TC ≈ 6.1:1; p = 0.0621, trend). Chow/Kd mice gained significantly less weight than chow-fed controls, whereas HFD/Kd mice exhibited weight gain comparable to HFD controls despite severe hepatic steatosis. This paradoxical phenotype suggests impaired metabolic feedback at the post-transcriptional level, in which compensatory upregulation of Hnf1a mRNA is insufficient to suppress lipid-associated genes such as Cd36, despite profound lipid overload; however, HNF1A protein levels were not directly measured in this study. Conclusion: Collectively, these findings identify Hnf1aos1 as a regulator of hepatic lipid homeostasis whose loss produces a phenotype consistent with inappropriate lipid accumulation during nutrient excess, without defining the underlying molecular mechanism. Our results support a role for Hnf1aos1 in shaping hepatic metabolic plasticity and provide insight into lncRNA-associated MASLD phenotypes. Full article

Nuclear factor kappa B (NF-κB) signaling plays a central role in inflammation, immunity, cell survival, and cancer progression. Its constitutive activation is frequently observed in breast cancer, contributing to tumor growth, treatment resistance, and metastasis. MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and may modulate NF-κB signaling in a subtype-specific or -independent manner. The aim of the study was to identify miRNAs that may potentially regulate the activity of genes associated with NF-κB signaling across five molecular subtypes of breast cancer in Polish women. Tumor and matched normal tissue samples were collected from 405 patients with five breast cancer subtypes: luminal A (n = 130), HER2-negative luminal B (n = 100), HER2-positive luminal B (n = 96), non-luminal HER2-positive (n = 36), and triple-negative breast cancer (TNBC, n = 43). Expression profile of selected NF-κB-related genes were evaluated using mRNA microarrays and RT-qPCR. Protein levels were assessed by ELISA. Candidate regulatory miRNAs were identified via miRNA microarrays and validated using the miRDB database. A consistent upregulation of MAP3K7, TAB2, TNFAIP3, CSNK2A1, BCL2L1, XIAP, CXCL2, and PLAU was observed across all subtypes, suggesting activation of canonical NF-κB signaling. Downregulation of specific miRNAs, miR-1297 and miR-30a (targeting MAP3K7), miR-134 (TAB2), miR-125b (TNFAIP3), and miR-4329 (XIAP), may contribute to this deregulation. For CSNK2A1, BCL2L1, CXCL2, and PLAU, no regulatory miRNAs meeting our criteria were identified. Our study reveals a subtype-independent activation of the canonical NF-κB signaling pathway in breast cancer, underpinned by consistent upregulation of key components (at both the transcript and protein levels. Dysregulation of specific miRNAs likely contributes to this altered gene expression. These findings suggest the presence of a common NF-κB-driven oncogenic program across molecular subtypes, with potential implications for developing miRNA-based therapeutic strategies targeting inflammation, survival signaling, and treatment resistance in breast cancer. Full article

Seasonal estrus limits sheep farming efficiency, making enhanced reproductive capacity and year-round estrus crucial for efficient breeding. GRM1 could modulates neuronal signals and stimulates neuron excitability. Our preliminary transcriptomic analysis of hypothalamic tissues from Kazakh ewes during nonbreeding season anestrus and nonbreeding season nutritionally induced estrus strongly suggested that GRM1 is a candidate gene that regulates estrus. The role of GRM1 in sheep estrus remains to be investigated. GRM1 expression was measured in hypothalamic tissues of Kazakh sheep during nonbreeding anestrus and nutritionally induced estrus via qPCR and immunohistochemistry. GRM1’s regulatory role in GnRH secretion and gene expression was studied in hypothalamic neurons via overexpression and RNAi. GnRH secretion changes were quantified by ELISA. GRM1 mRNA expression was significantly increased in the hypothalamus of estrous Kazakh sheep, as confirmed by immunohistochemical staining. The results of hypothalamic neuron experiments revealed that the expression of GRM1 was significantly upregulated after overexpression, which affected the expression of GnAQ, ITPR1, PLCB1 and PRKCB and ultimately promoted the secretion of GnRH. The expression of GnRH decreased after the interference in GRM1 expression. GRM1 modulates the secretion of GnRH in the hypothalamic nerve cells of Kazakh sheep through the glutamatergic synapse–calcium signaling pathway. Full article

Listeria monocytogenes (Lm) is an important zoonotic foodborne pathogen that causes severe rhombencephalitis in ruminants. The trigeminal ganglion is a critical node for Lm invasion of the central nervous system via neural pathways. However, the roles of key virulence factors InlA, InlB, and LLO from ovine-derived Lm in trigeminal ganglion neuron infection remain unclear. In this study, LM90SB2, an ovine-derived Lm strain isolated from a sheep with encephalitis in Xinjiang, China, was used as the wild type, and its ΔInlAB double-gene deletion and ΔInlABO triple-gene deletion mutants were constructed. Primary mouse trigeminal ganglion cells (TGCs) were infected with these strains, and cell-association and invasion assays, bacterial colonization analysis, cell scratch tests, Western blotting, and qRT-PCR were performed to explore the effects of InlA, InlB, and LLO on Lm infection of TGCs and their regulatory roles in host adhesion molecules N-cadherin and NCAM1. The results showed that the wild-type LM90SB2 had significantly stronger cell-association, invasion, and colonization abilities in TGCs than the ΔInlAB and ΔInlABO mutants (p < 0.01 or p < 0.0001). LM90SB2 infection significantly upregulated the mRNA and protein expression levels of N-cadherin and NCAM1 in TGCs and enhanced TGC migration, while these effects were gradually attenuated with the sequential deletion of InlA, InlB and LLO. This study clarifies the synergistic roles of InlA, InlB, and LLO in mediating the infection of trigeminal ganglion neurons by ovine-derived Lm and reveals the molecular mechanism by which Lm promotes neural invasion by regulating the expression of host cell adhesion molecules. Our findings provide important experimental data for elucidating the neural invasion pathway of Lm in ruminants and lay a theoretical foundation for the development of targeted prevention and control strategies for ruminant listeriosis in veterinary clinical practices. Full article

Obstetrical diseases are complications associated with pregnancy or childbirth that can cause maternal sequelae and fetal complications. Among them, preeclampsia (PE) and preterm labor (PTL) are major causes of premature birth and are associated with an increased risk of cerebral palsy, developmental delay, and hearing impairment in infants. However, reliable diagnostic markers and therapeutic strategies for obstetrical diseases remain limited. The aim of this study was to investigate genes associated with obstetrical diseases and to evaluate the correlation between phosphocholine cytidylyltransferase 1 beta (PCYT1B) and miRNAs targeting PCYT1B for diagnostic analysis in PE and PTL. Using miRNA array analysis and luciferase assays, we identified PCYT1B, a key enzyme involved in phosphocholine metabolism in reproductive tissues, together with several candidate miRNAs targeting PCYT1B, including miR-3065-3p, miR-4660, miR-6752-5p, miR-6842-5p and miR-7110-5p. qRT-PCR analysis revealed a significant correlation between PCYT1B and these miRNAs in placental tissues from patients with PE and PTL (p < 0.05). Immunofluorescence staining further demonstrated that PCYT1B was localized in the syncytiotrophoblast layer of placental tissues, and its protein expression was consistent with mRNA expression levels. To investigate the functional role of these miRNAs, trophoblast cells were treated with miRNA mimics and inhibitors. These treatments significantly altered trophoblast invasion capacity and regulated the expression of migration-related genes, including RhoA, Rac1 and ROCK. Collectively, our findings suggest that miRNAs targeting PCYT1B may regulate trophoblast function and may play a key role in placental development and obstetrical diseases. These results indicate that PCYT1B and its regulatory miRNAs could serve as potential biomarkers for PE and PTL and may provide insights into the development of miRNA-based diagnostic strategies. Full article

Fine particulate matter (PM_2.5) can directly impact pulmonary epithelial cells, resulting in lung injury. While it is known that PM_2.5 can alter the expression profile of microRNAs in the lung, its specific role in damaging pulmonary epithelial cells remains unclear. This study, therefore, employed RT-qPCR, Western blotting, and dual luciferase reporter assays to investigate the regulatory role of microRNAs in PM_2.5-induced cellular damage. PM_2.5 exposure induces oxidative stress, autophagy, and ferroptosis in rat lung alveolar epithelial cells (RLE-6TN). Further functional rescue experiments confirm that the ferroptosis-specific inhibitor Fer-1 can block PM_2.5-induced ferroptosis. Bioinformatics analysis and validation indicate that miR-212-5p plays a crucial role by negatively regulating RASSF1 through targeted inhibition. Overexpression of miR-212-5p activates the PI3K/AKT signaling pathway, thereby promoting autophagy and ferroptosis. However, when the expression of both miR-212-5p and RASSF1 is suppressed, PM_2.5-induced autophagy and ferroptosis are significantly alleviated by inhibiting the PI3K/AKT/mTOR signaling pathway. Rescue validation experiments demonstrated that, under PM_2.5 exposure combined with RASSF1 overexpression, miR-212-5p exacerbates the aforementioned cellular damage process. This study reveals that miR-212-5p regulates autophagy and ferroptosis by targeting RASSF1. These findings provide a multi-target intervention strategy for PM_2.5-related lung diseases. Full article

Uridine diphosphate glucuronosyltransferases (UGTs) are critical phase II detoxification enzymes; however, their mutational landscape and protective roles against chemical carcinogenesis in hepatocellular carcinoma (HCC) remain poorly defined. Here, targeted sequencing of ten liver-enriched UGT genes in 38 paired tissues from a Chinese HCC cohort revealed striking mutation frequencies in UGT2B15 (44.74%), UGT2B10 (36.84%), and UGT2B17 (26.32%). This genomic instability was accompanied by a profound downregulation of UGT2B15 mRNA (9.02-fold decrease, p < 0.001) and protein levels (Z-score = 2.32, p = 0.0093) in tumors, with higher UGT2B15 expression correlating with improved overall survival in TCGA cohorts (HR = 1.724, p = 0.012). Mechanistically, we identified the androgen receptor (AR) as a direct transcriptional regulator of UGT2B15 and UGT2B17, with dihydrotestosterone (DHT) inducing dose-dependent increases in their expression, thereby linking endocrine signaling to hepatic detoxification. Transcriptomic profiling following UGT2B15 knockdown in HCC cells revealed a significant enrichment in chemical carcinogenesis-related pathways. Crucially, UGT2B15 deficiency severely exacerbated carbon tetrachloride (CCl₄)- and ethanol-induced hepatotoxicity both in vitro and in vivo. Our study uncovers a profound impairment of UGT-mediated detoxification in HCC and establishes the AR–UGT2B15 axis as a critical barrier against chemical-induced liver injury, highlighting its potential as a chemopreventive target in carcinogen-exposed populations. Full article

Background/Objectives: Calcium–phosphorus metabolism is critical for skeletal development in weaned piglets. This study evaluated the effects of dietary 25-hydroxyvitamin D₃ (25-OH-VD₃) in combination with phytase and probiotics on mineral metabolism, bone development, and related molecular mechanisms in weaned piglets. Methods: Sixty 28-day-old weaned piglets (7.1 ± 1.30 kg) were randomly assigned to four dietary treatments for 31 days (including 3 days of acclimation): CON (basal diet + 50 µg/kg 25-OH-VD₃), HI (CON + 50 mg/kg phytase), CY (CON +10 mg/kg probiotics), HICY (CON + 50 mg/kg phytase + 10 mg/kg probiotics). Apparent calcium digestibility, serum biochemical indices, bone mineral density (BMD), and mRNA and protein expression of calcium–phosphorus transport- and metabolism-related genes in jejunal mucosa and kidney were assessed. Results: Compared with CON, piglets in the HI, CY, and HICY groups showed higher apparent calcium digestibility (p < 0.05). Serum transforming growth factor-β was elevated in CY and HICY (p < 0.05). HI enhanced metatarsal and toe BMD (p < 0.05) and upregulated jejunal solute carrier family 34, member 2 (SLC34A2) and SLC34A3 mRNA expression (p < 0.05). In contrast, HICY reduced mRNA expression of transient receptor potential cation channel subfamily V member 6 and calcium-binding protein D28k, as well as of calcium-binding protein D9k and cytochrome P450 27B1 in the kidney (p < 0.05). Renal calcium-sensing receptor protein abundance increased in CY (p < 0.05). Conclusions: Supplementation of 25-OH-VD₃ with phytase and/or probiotics improved calcium utilization and modulated key transport pathways, contributing to enhanced bone development in weaned piglets. These findings highlight coordinated nutritional regulation of mineral metabolism during early post-weaning growth. Full article

To investigate the response mechanism of cold-resistant Enshi black pig breeds under cold stress, nine Enshi black pigs were randomly divided into three groups with three pigs in each: a control group (18 ± 2 °C for 58 d), a cold-stress-acclimated group (3 to 8 °C to −17 to −21 °C for 58 d), and an acute cold stress group (−17 to −21 °C for 3 d). RNA-seq technology was used to analyze mRNA and lncRNA expression patterns in subcutaneous adipose tissue under cold stress. The results showed that, under acute cold stress, many metabolic pathways were activated, including those involved in rapid energy supply (e.g., the citric acid cycle/TCA cycle, fatty acid degradation and metabolism, and glycolysis/gluconeogenesis), signal transduction pathways (e.g., PI3K Akt, MAPK, PPAR, HIF-1, mTOR, and FoxO), and immune and cellular homeostasis pathways (chemokine signaling pathway, T cell receptor signaling, Toll-like receptor signaling, and apoptosis and autophagy regulation). Under cold stress acclimation, metabolic regulatory pathways (e.g., AMPK, mTOR, FoxO, HIF-1, glycolysis/gluconeogenesis, and fatty acid degradation), immune and inflammatory regulatory pathways (Toll-like receptors, NOD like receptors, and T/B cell receptor signaling pathways), and signal transduction and cell homeostasis pathways (MAPK, PI3K Akt, NF-κB, Notch signaling pathways, apoptosis, and autophagy regulation) were continuously activated to ensure the stability of adipose tissue structure and function. Acute cold stress activated more pathways than cold stress acclimation, but both led to significant changes in energy metabolism. The results identified the molecular regulatory mechanisms of adipose tissue under cold stress, providing a basis for the subsequent breeding of new cold-resistant pig breeds. Full article

Background: An accurate, sensitive, and robust potency assay is essential for the quality control of mRNA drug substances, which are characterized by complex manufacturing processes, intricate molecular structures, and high susceptibility to degradation. Currently, mRNA vaccine manufacturers use a variety of biological potency assays, often without systematic method development or rigorous evaluation. As a result, these assays may lack sufficient accuracy and robustness, making it difficult to reliably distinguish mRNA drug substance samples with different potency levels. Therefore, there is a need for a standardized, robust, and reliable potency assay for the evaluation of mRNA drug substance samples across a range of potencies. Methods: In this study, we developed a cell-based relative potency assay for a respiratory syncytial virus (RSV) mRNA drug substance encoding an engineered prefusion (PreF) form of the RSV type A (RSV-A) F protein, a recognized target for RSV vaccine development. The RSV mRNA drug substance was complexed with transfection reagents and introduced into cells in vitro to enable expression of the RSV-A PreF protein, which was then quantified using a double-antibody sandwich ELISA. Results: Systematic optimization showed that cell line, cell density, transfection reagent, mRNA-to-transfection reagent ratios, and transfection duration all influenced assay performance. Under optimized conditions, the assay demonstrated acceptable accuracy and precision, with relative bias values ranging from −25% to 13% across the potency range of 44~156%, measured-to-expected ratios within 0.8~1.2, and relative standard deviations of 18% and 16% for intra- and inter-assay precision, respectively. Furthermore, the optimized potency assay effectively distinguished mRNA drug substance samples with varying potency levels. Conclusions: This study provides a useful functional complement to physicochemical characterization and supports quality control and batch-to-batch consistency of RSV mRNA drug substances. In addition, the development strategy may also serve as a useful reference for the establishment of in vitro potency assays for other mRNA drug substances. Full article

Regulation of all-trans retinoic acid (ATRA) signaling is crucial to early embryonic development. Embryonic stem (ES) cell-derived gastruloids mimic normal development in response to the Wnt/β-catenin agonist CHIR9901, and this study has examined the importance of the activities of RAR (retinoic acid receptor) α and γ to gastruloid development. Expression of retinoic acid receptor (RAR)γ within developing gastruloids was spatially restricted to primitive cells that co-expressed ES cell and early progenitor cell markers, i.e., Nanog, Sox2, and Oct4. In contrast, RARα expression was ubiquitous. mRNAs for the key enzymes involved in ATRA synthesis (Aldh1a2) and degradation (Cyp26a1) were not seen in cells that expressed RARγ. Treatment of ES cell-derived gastruloids with physiologically relevant (10 nM) levels of ATRA or with a highly selective RARγ agonist blocked normal developmental processes, preventing symmetry-breaking and axial elongation. This was not seen following treatments with an RARα agonist, where there was a tendency for enhanced axial elongation. Brachyury (TBXT) immuno-positive cells localized in the posterior end of elongated gastruloids in control- and RARα agonist-treated cultures, with Sox2 immuno-positive cells seen more widely, whilst both TBXT and Sox2 immuno-positive cells were randomly distributed throughout ATRA- and RARγ agonist-treated gastruloids. Concurrent treatment of gastruloids with 10 nM ATRA and 100 nM of an RARγ antagonist partially abrogated the ATRA-mediated block to axial elongation. Conversely, 10 nM RARγ antagonist treatments were associated with the formation of multi-axis gastruloid elongations, with comparatively little effect seen after treatments with an RARα antagonist. These findings reveal that RARγ plays a crucial role in the development of embryonic tissues. Full article

Oral squamous cell carcinoma (OSCC) remains a major cause of cancer-related morbidity and mortality, and reliable biomarkers for early diagnosis and risk stratification are still lacking. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) can regulate gene expression through competing endogenous RNA (ceRNA) interactions, but OSCC-specific ceRNA axes with clinical relevance are still poorly defined. We integrated lncRNA, miRNA, and mRNA expression data from six OSCC-related datasets in the Gene Expression Omnibus with in silico interaction predictions to construct an OSCC-focused ceRNA network and examine its association with survival. The resulting network comprised 8 mRNAs, 22 miRNAs, and 12 lncRNAs. Within this network, we identified a previously unrecognized XIST–miRNA–ZNF662 axis that has not been characterized in OSCC. ZNF662 was consistently downregulated in tumors, and higher ZNF662 expression was associated with improved survival in an independent head and neck squamous cell carcinoma cohort. Components of the XIST–miRNA–ZNF662 axis also showed excellent diagnostic performance for distinguishing OSCC from normal samples across (Gene Expression Omnibus) GEO datasets, highlighting a ceRNA module with promising diagnostic and prognostic potential that could be explored further in non-invasive biofluids. Full article

Glucose oxidase (GOD) is a natural enzyme with antioxidant and antimicrobial properties but its effects on sows remain insufficient. This study investigated the effects of dietary GOD supplementation during gestation on inflammatory response, antioxidant capacity, immune function, and gut microbiota of farrowing sows. Twenty-four primiparous sows were randomly assigned to two groups and fed a basal diet or a basal diet supplemented with GOD (300 mg/kg diet) from gestation day 30 to farrowing. GOD supplementation significantly increased triglyceride, superoxide dismutase, and immunoglobulin M levels (p < 0.05), and significantly decreased alanine aminotransferase and interleukin-6 levels in serum (p < 0.05); significantly reduced placental interleukin-1β, malondialdehyde and tumor necrosis factor-α concentrations and NF-κB gene expression (p < 0.05), and elevated glutathione peroxidase activity and relative mRNA expressions of Nrf2, HO-1, GPX1 and SOD2 (p < 0.05). Moreover, GOD supplementation altered the fecal microbial community structure (p < 0.05), significantly reducing Clostridium, dgaA-11_gut_group, Bacteroides, and Prevotellaceae_NK3B31_group abundance (p < 0.05), while enriching Lachnospira, unclassified_f_Erysipelotrichiaceae, and Anaerostipes (p < 0.05). Collectively, 300 mg/kg glucose oxidase supplementation during mid-to-late gestation improved the health status of farrowing sows by improving nutrient utilization, immune function and antioxidant capacity, and altering fecal microbial structure and relative abundances. Full article

Enteroviruses are positive-sense single-stranded RNA viruses and common pathogens that are responsible for diverse public health diseases. To facilitate the study of the virus biology and pathogenesis of enterovirus, we developed a rapid method for construction of the enteroviral cDNA clones including enterovirus A71 (EV-A71) and coxsackievirus B5 (CVB5). As described for EV-A71, the full-length cDNA of CVB5 was amplified by long-distance PCR and cloned into a T7 promoter-containing plasmid using directional seamless cloning technology. The virus was successfully rescued by single transfection into cells stably expressing T7 polymerase and exhibited characteristics similar to the parental virus. Next, through systematic construction and the optimization of the EV-A71 and CVB5 reporter viruses, we successfully generated two novel reporter virus panels with high virus titers, rapid replication, and relatively stable genetic inheritance across passages using the new fluorescence proteins mScarlet3-H and the smallest miRFP670nano3. Analysis of critical determinants for the reporter virus construction revealed that reporter gene sizes, genomic insertion sites, and the usage of protease recognition sites are crucial parameters. The EV-A71 and CVB5 reporter viruses enable antiviral drug evaluation, as demonstrated by our identification of gemcitabine as a broad-spectrum inhibitor of both viruses. These systems also facilitate the functional interrogation of host factors, exemplified by our discovery that METTL3 promotes EV-A71 and CVB5 replication. These reverse genetic tools, including infectious cDNA clones and reporter viruses, will advance basic enterovirus biology and accelerate antiviral drug discovery. Full article

Amyloid-β (Aβ) protein, a cleavage product of the amyloid precursor protein (APP), is the main component of neuritic plaques in Alzheimer’s disease (AD), and its accumulation has been considered as the molecular driver of Alzheimer’s pathogenesis. Aβ has been a primary target for therapy since the amyloid cascade theory was put forth, with methods designed to prevent the generation of Aβ. The APP 5′-untranslated region (UTR) mRNA encodes a functional structured iron-responsive element (IRE) that represents a potential target for small molecule inhibitors as an anti-amyloid therapy for AD. Here, we offer a comprehensive strategy that uses RNA-targeted binding to inhibit APP translation. The IRE family is among the few 3-D mRNA regulatory elements with a known 3-D structure. Accordingly, we exploit these structural and functional characteristics as our strategy to target APP IRE structured mRNA to identify anti-amyloid drugs. The mRNA encoding proteins involved in iron metabolism are regulated by this family of similar nucleotide sequences. Post-transcriptional control of cytoplasmic mRNA is a rapidly developing area of biomedicine. Across animals, evolutionarily conserved IRE mRNAs serve as a model system for 3-D mRNAs. IRE mRNAs have shown great promise for chemical manipulation of mRNA and protein expression in biological systems by yielding “proof of principle” data for small molecules targeting mRNA structures. A novel approach to identifying RNA-directed therapeutics to regulate APP expression and Aβ-peptide generation for AD treatments is exemplified by APP 5′-UTR-directed small molecule inhibitors. Full article