Search Results (905)

(1) Background: Seasonal breeding constraints represent a long-standing barrier to efficient sheep production worldwide. Small Tail Han sheep, a Chinese indigenous breed that exhibits year-round estrus independent of photoperiod, offers a unique natural model for investigating the regulatory mechanisms underlying estrus cyclicity—yet the molecular basis of this remarkable trait remains largely unexplored. This study therefore aimed to compare ovarian whole-transcriptome profiles between Small Tail Han sheep (year-round estrus) and Ujumqin sheep (seasonal estrus) during estrus and diestrus. Specifically, we sought to characterize breed-specific transcriptional response strategies, identify key miRNAs associated with continuous estrus, and construct ceRNA networks that mediate ovarian estrous cycle regulation. (2) Methods: To address these objectives, we profiled ovarian transcriptomes of Small Tail Han sheep (year-round estrus) and Ujumqin sheep (seasonal estrus) during both estrus and diestrus (12 samples total) using whole-transcriptome sequencing, systematically characterizing the expression landscapes of mRNAs, miRNAs, lncRNAs and circRNAs. Through differential expression analysis, GO/KEGG functional enrichment, multi-tiered miRNA screening, and ceRNA network construction, we compared the transcriptional response strategies between the two breeds. (3) Results: Our findings revealed two unexpected and divergent patterns. Small Tail Han sheep showed 1881 differentially expressed mRNAs between estrus and diestrus, 82-fold more than the 23 in Ujumqin sheep, suggesting a broader mRNA-level transcriptional response in the year-round estrus breed. circRNAs were predominantly up-regulated (80–85%) in both breeds, while mRNAs and lncRNAs were predominantly down-regulated, uncovering a conserved “circRNA-up, mRNA-down” regulatory logic across breeds. Multi-tiered screening identified 38 “brake-releaser” miRNAs (high in diestrus, down-regulated in estrus) and 21 “accelerator” miRNAs (low in diestrus, up-regulated in estrus), which together constitute a “de-repression–activation” dual regulatory mode. ceRNA network analysis further revealed a clear functional stratification: conserved core miRNAs targeted “sensor” pathways (cAMP, PI3K-Akt, MAPK), whereas accelerator miRNAs specifically targeted “executor” pathways (estrogen signaling, progesterone-mediated oocyte maturation, Hippo signaling). Network topology analysis pinpointed miR-199a-5p, miR-204-3p and miR-199b as the core hubs bridging the two regulatory modules. (4) Conclusions: Taken together, our findings redefine the molecular basis of year-round estrus—moving beyond single-gene mutations or overexpression to an enhanced global transcriptomic responsiveness of the ovary. This work provides a fresh conceptual framework for understanding the divergence of reproductive strategies in sheep and offers actionable candidate targets for genetic improvement of continuous estrus traits. Full article

Chronic myeloid leukemia (CML) is a type of bone marrow cancer characterized by the uncontrolled proliferation of myeloid cells. MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in the post-transcriptional regulation of gene expression. This study aims to examine the association between miR-33a-5p and Ankyrin 3 (ANK3) in CML and to investigate the regulatory mechanisms of miR-33a-5p in the progression of this disease. mRNA expression profiles were obtained from the GSE100026 dataset within the Gene Expression Omnibus (GEO) repository. Quantitative real-time polymerase chain reaction (RT-qPCR) was conducted to assess the expression levels of miR-33a-5p and ANK3. To investigate the regulatory mechanism of miR-33a-5p/ANK3, various databases such as miRNet, miRDIP, TargetScan, BioGRID, and CancerSEA were utilized. The expression of miR-33a-5p was markedly elevated, while ANK3 expression was significantly reduced. Furthermore, pathway clustering and functional assessments of ANK3 demonstrated its involvement in regulating the cell cycle and apoptosis. The findings identify an inverse expression pattern between miR-33a-5p and ANK3 across the two cell models and support a candidate regulatory relationship that warrants direct functional validation. This relationship may merit further investigation as a potential molecular target in CML. Full article

Spatial heterogeneity is increasingly recognized as a major determinant of molecular responses in inflammatory bowel disease, yet molecular analyses in dextran sulfate sodium (DSS)-induced colitis commonly rely on whole-colon measurements that may obscure localized biological processes. We investigated how colon layer, histological phenotype, and colitis phase influence RNA expression by combining archived formalin-fixed paraffin-embedded (FFPE) colon tissue, histology-guided laser microdissection (LMD), targeted RT-qPCR, and multivariate modelling. Expression of five mRNAs (Tnf, Tnfr1, Tnfr2, Duox2, and iNos) and three miRNAs (miR-223-3p, miR-181a-5p, and miR-3473b) was analyzed in whole-colon sections, individual colon-wall layers, and histologically defined mucosal microenvironments. RNA expression varied according to anatomical layer, histological phenotype, and colitis phase, with the most pronounced molecular changes observed in erosive mucosa. Although several RNAs showed similar overall trends, whole-section measurements did not consistently reflect the magnitude or spatial distribution of RNA expression observed in individual layers and histological microenvironments. Multivariate modelling further revealed context-dependent interrelationships among the analyzed mRNAs and miRNAs and enabled these molecular patterns to be evaluated in relation to histological, anatomical, and temporal variables. These findings demonstrate that spatial and histological context can substantially influence the interpretation of targeted molecular measurements in DSS colitis and that whole-colon analysis may miss spatially restricted molecular changes. Histology-guided analysis of archived FFPE tissue, combined with multivariate modelling, therefore provides a complementary approach for resolving tissue heterogeneity and extracting additional biological information from existing animal tissue repositories, potentially reducing the need for additional animal experiments when appropriate archived material is available. Full article

Background/Objectives: Tuberculosis is a risk factor for lung cancer. Previous studies have demonstrated that miRNAs derived from tuberculous pleural effusion (TPE) exosomes are involved in lung cancer progression. This study aimed to identify potential miRNA–mRNA regulatory networks associated with TPE-derived exosome exposure in the context of lung cancer. Methods: Exploratory differential expression analysis was performed using RNA-seq data from TPE-derived exosomes to select candidate miRNAs showing differential expression patterns. In an in vivo mouse xenograft tumor model, TPE-derived exosomes were administered intratumorally, followed by RNA-seq analysis to select candidate genes showing differential expression patterns. Predicted target genes of the candidate miRNAs were then overlapped with the candidate genes from the xenograft analysis to obtain overlapping common genes (OCGs). The regulation of these genes by candidate miRNAs was validated through in vitro experiments, and a miRNA-mRNA network was constructed using Cytoscape. Results: Exploratory analyses suggested differential expression patterns in 27 candidate miRNAs (19 showing increased and 8 showing decreased expression) in TPE-derived exosomes and 2340 candidate genes in xenograft tissues. A total of 4715 and 700 target genes were predicted for candidate miRNAs using miRDB and miRTarBase, respectively. Following a stepwise filtering strategy, five candidate target genes (RRAS2, RAC1, RAP1B, NOTCH2, and SMARCA4) were prioritized from the overlapping common genes (OCGs). The seven candidate miRNAs predicted to regulate these genes were subsequently used to prioritize a candidate 7-miRNA–5-mRNA regulatory network for further investigation. Conclusions: This study prioritized a candidate miRNA–mRNA regulatory network associated with TPE-derived exosome exposure in lung cancer models, providing a basis for further investigation in larger clinical cohorts and more physiologically relevant models. Full article

Background: Osteoarthritis (OA) is a chronic whole-joint disease involving extracellular matrix remodeling, inflammatory signaling, immune–stromal interactions, and sensory-associated processes. MicroRNAs (miRNAs) may contribute to post-transcriptional regulation within these interconnected molecular domains, but concurrent mRNA and miRNA expression patterns remain insufficiently characterized in naturally occurring canine OA. Methods: A total of 12 formalin-fixed, paraffin-embedded (FFPE) canine tissue specimens were classified as osteoarthritic joint tissues (OA; n = 7) and control joint tissues without radiographic or histopathological evidence of OA (C-JT; n = 5). RT-qPCR was used to quantify 17 selected mRNAs involved in extracellular matrix remodeling, chronic inflammatory signaling, immune–stromal interactions, and neuroinflammatory and sensory-associated processes, together with five miR-30 family members. Statistical comparisons were performed on ΔCq values using two-sided unpaired Welch’s t-tests followed by Benjamini–Hochberg false discovery rate correction. Results: Compared with C-JT, OA tissues showed significantly higher expression of ADAMTS5, MMP2, MMP13, COL2A1, SPARC, HP, VEGFA, IGFBP6, and ASIC3 after false discovery rate correction. MMP9, IL1B, IL1R1, IL1RAP, JAK2, TYK2, NGFB, and NTRK1 did not differ significantly between the two joint-tissue groups. MiR-30b was significantly downregulated in OA, whereas miR-30c, miR-30d, and miR-30e were significantly upregulated; miR-30a did not differ significantly. Exploratory database-supported miRNA–gene connectivity was used for candidate prioritization but was not interpreted as evidence of direct regulation in canine joint tissue. Conclusions: Canine osteoarthritic capsulosynovial tissues exhibit concurrent alterations in selected mRNAs and miR-30 family members across matrix-remodeling, immune–stromal, and sensory-associated molecular domains. These findings identify OA-associated expression patterns but do not establish pathway activation, direct miRNA–mRNA regulation, or disease progression. They provide a focused molecular basis for future mechanistic studies of post-transcriptional regulation in naturally occurring canine OA. Full article

Neoadjuvant chemotherapy (NAC) is the standard of care for locally advanced breast cancer, yet the molecular predictors of pathological response remain incompletely defined, particularly regarding microRNA (miRNA) dynamics. We investigated paired pre- and post-NAC miRNA expression profiles in relation to molecular subtype, residual cancer burden (RCB), and clinical timing parameters. Seven patients with invasive breast cancer (Luminal A n = 3, Luminal B n = 1, TNBC n = 2, HER2+ n = 1) who received NAC (AC-T or TCHP) were included in this pilot study. Small-RNA sequencing (NovaSeq X Plus, CeGaT GmbH, project S17293) was performed on 14 FFPE specimens (7 pre-NAC core needle biopsies, 7 post-NAC surgical specimens). Differential expression analysis used the paired Wilcoxon signed-rank test with Benjamini–Hochberg correction. Spearman correlations assessed associations between miRNA expression, RCB score, and clinical timing intervals. Candidate miRNAs were subsequently annotated using experimentally validated miRNA–target interactions. After filtering (≥ three counts in ≥ three samples), 759 miRNAs were analysed. No miRNA reached strict significance (padj < 0.05, |log₂FC| > 1.0) after multiple testing correction, consistent with the limited statistical power (n = 7). Under exploratory criteria (p < 0.10, |log₂FC| > 0.5), 156 candidate miRNAs were identified: 70 upregulated and 86 downregulated post-NAC. Leading candidates included hsa-miR-139-3p (+2.60), hsa-miR-139-5p (+2.36), and hsa-miR-1323 (+1.55) as upregulated, and hsa-miR-429 (−2.53), hsa-miR-141-3p (−1.79), and hsa-miR-1277-5p (−1.25) as downregulated post-NAC. The single patient achieving the lowest residual disease burden (P3, Luminal B, RCB-I, score 1.32) displayed a distinct pre-treatment miRNA profile, separating from all other pre-NAC specimens on principal component analysis and characterised by higher baseline hsa-miR-139-3p/-5p and lower baseline hsa-miR-429 and hsa-miR-141-3p expression, suggesting that baseline miRNA expression patterns may contribute to differential chemotherapy response. RCB score showed a non-significant positive trend with post-NAC Ki-67 (ρ = +0.71, p = 0.07). This pilot study identifies NAC-modulated candidate miRNAs in breast cancer and establishes a paired FFPE-based small-RNA-sequencing workflow applicable in routine clinical settings. The distinct pre-treatment profile of the single best responder generates the testable hypothesis that baseline expression of tumour suppressor miRNAs of the miR-139 family, together with low miR-200-family expression, may track chemosensitivity. As no candidate reached statistical significance after multiple testing correction and none has been validated in an independent cohort or by an orthogonal method, all findings are exploratory and hypothesis-generating. The results support larger prospective validation studies examining miRNA signatures as predictive biomarkers of NAC response across breast cancer molecular subtypes. Full article

Grafting profoundly influences fruit tree performance, yet the molecular mechanisms underlying rootstock–scion interactions in mulberry (Morus multicaulis) remain poorly understood. To investigate the regulatory networks linking grafting to scion physiology, we performed an integrated analysis combining small RNA sequencing, transcriptome-based KEGG pathway assessment, and qRT-PCR validation of key target genes across five rootstock–scion combinations. High-throughput miRNA profiling of phloem tissues identified 71 conserved and 156 novel miRNAs, which exhibited distinct, genotype-dependent expression patterns. Recurrent activation of nutrient- and stress-responsive families (e.g., miR399, miR397, and miR395) was observed. Target prediction and functional enrichment analyses revealed that these miRNAs likely regulate pathways related to metabolic processes, hormone signaling, cell wall modification, and stress responses. The expression trends of their predicted target genes—including TCP4, Laccase-3, and ATP sulfurylase 1—were subsequently confirmed by qRT-PCR, revealing significant rootstock-specific regulation. Collectively, this study establishes a reference framework for elucidating the molecular mechanisms underlying scion biological processes in Morus species, which is of great significance for improving fruit quality, enhancing abiotic and biotic stress resistance, and boosting long-term planting productivity. Full article

A-to-I RNA editing is increasingly recognized as a regulator of fungal development and pathogenesis, yet its extent and lifecycle dependence remain poorly characterized in basidiomycetes. Microbotryum superbum (MvSup), M. intermedium (MI), and M. lychnidis-dioicae (MVLG) are members of the M. violaceum fungal complex. Each species infects specific host plant species, resulting in commonly anther-smut disease. The lifecycle of these basidiomycete fungi includes the haploid, mating, and infection stages. RNA editing is a post-transcriptional process where adenosine (A) is converted to inosine (I) by adenosine deaminase enzymes, and such modifications to RNAs may lead to synonymous and nonsynonymous codon changes, thereby altering protein function. Here, we compared A-to-I editing across the haploid, mating, and infection stages of these three related species to determine how editing patterns vary with lifecycle stage and among closely related fungal pathogens. The a2 haploid strain of MI had fewer editing sites compared to other haploid strains. The predicted codon/amino acid changes in each haploid strain across the three species indicated three primary types of resulting amino acid substitutions that were common to both of the mating-type strains across the three species: threonine to alanine, lysine to glutamic acid, and valine to alanine. During the mating stage of MvSup, a synonymous codon change was found in a mitogen-activated protein kinase domain-containing protein within the protein’s conserved region. Gene expression analysis revealed that certain genes, uniquely edited during the mating stage of MvSup, tend to be upregulated in the haploid stage but downregulated during mating, and vice versa. Research on RNA editing in basidiomycetes is relatively new. RNA editing mechanisms in fungi have been implicated in fungal pathogenesis, although the exact roles and implications remain unclear. Additional research will help us understand the functional significance of this apparently ubiquitous process in several members of the Microbotryum fungal complex, with possible ramifications more generally in fungi. Full article

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality owing to late diagnosis, aggressive tumor biology, and limited clinically useful early diagnostic and prognostic biomarkers. This study provides an exploratory evaluation of the expression profile, survival associations, co-expressed genes, functional association networks, and regulatory relationships of MEAK7 (KIAA1609/TLDC1) in pancreatic cancer using multi-platform bioinformatic approaches. We conducted a comprehensive multi-platform bioinformatic analysis using multiple publicly available datasets and platforms, including TCGA/GTEx, GEO, GEPIA3, HPA, CPTAC/UALCAN, TNMplot, Kaplan–Meier Plotter, DoSurvive, TIMER 3.0, STRING, TargetScan, miRDB, ENCORI and lncRNADisease. Gene expression patterns, survival associations, immune cell infiltration and regulatory non-coding RNA networks were systematically investigated. A STRING-derived protein functional association network was evaluated. MEAK7 expression patterns were additionally assessed in external GEO datasets, including GSE62165, GSE71729, and GSE183795. In summary, MEAK7 expression was elevated in pancreatic cancer compared with normal pancreatic tissues, was detectably expressed across multiple pancreatic cancer cell lines, and was increased in patient-derived PDAC samples. ROC analysis showed that MEAK7 expression discriminated PDAC tumor tissues from adjacent non-tumor pancreatic tissues, with an AUC of 0.795 (95% CI: 0.719–0.871) indicating exploratory transcriptomic discrimination rather than clinical diagnostic performance. Exploratory survival analyses showed that elevated MEAK7 expression was associated with unfavorable overall survival and disease-free outcomes, while multivariable Cox analyses showed that this association persisted after adjustment for the covariates available within the analyzed dataset. GEO-based analyses supported increased MEAK7 expression in early-stage pancreatic cancer samples, although metastatic expression patterns varied across datasets. Correlation and interaction analyses revealed positive associations between MEAK7 and PDAC-related genes, including CTTN, CDCP1, SMARCA4, SFN, and PALB2, as well as high-confidence STRING functional associations with V-ATPase components and RNASEK. Furthermore, MEAK7 was positively correlated with lncRNA UCA1 and negatively correlated with miR-582-5p. Collectively, these findings identify reproducible expression and survival-associated patterns involving MEAK7 across the analyzed datasets and provide a hypothesis-generating framework for further investigation of its potential biological role in PDAC. Experimental studies and prospectively characterized patient cohorts are required before any clinical, prognostic, diagnostic, or therapeutic implications can be established. Full article

Severe coronavirus disease 2019 (COVID-19) is characterized by acute immune dysregulation, with monocytes playing a central role in driving inflammation and disease severity. However, the transcriptional and post-transcriptional regulatory mechanisms underlying monocyte dysfunction in severe COVID-19 remain unexplored. In the study, an integrative analysis of paired bulk RNA-seq and miRNA-seq datasets was performed together with independent single-cell RNA-seq (scRNA-seq) data from convalescent individuals with a history of ICU or non-ICU COVID-19. Pooled cell proportions descriptively indicated a higher proportion of classical monocytes and lower proportions of non-classical monocytes, B cells and dendritic cells in individuals with a history of ICU disease; however, none of these differences was statistically significant in patient-level analyses after multiple-testing correction. Using the miRSCAPE framework, miRNA expression was inferred at single-cell resolution and identified distinct cluster-specific inferred miRNA expression patterns. Differential expression analysis of classical monocyte populations identified 284 nominally significant differentially expressed genes between convalescent ICU and non-ICU samples. Integration of miRNA-mRNA correlation analysis with experimentally validated interactions and independent assessment highlighted a focused regulatory network centered on ZMAT3, RHOB and HLA-DQA1. Host–pathogen interaction analysis identified database-supported SARS-CoV-2-host interactions involving ORF3a-RHOB and nucleoprotein-RHPN2, with additional host–host interactions connecting RHPN2, HLA-C and HLA-DQA1. Collectively, these findings provide a computational framework for investigating inferred miRNA associations of monocyte inflammatory pathways associated with prior COVID-19 severity and highlight regulatory interactions that warrant further experimental validation. Full article

Pleurotus ostreatus is an important edible mushroom, and its growth and development are affected by genetic and epigenetic mechanisms. miRNAs play an important role in fungal epigenetic mechanisms. In previous studies, Po-3KAT was identified as a candidate target of Po-milR-1, and its product was a substrate for ergosterol synthesis. However, the potential relationship between Po-milR-1 and Po-3KAT has not been studied in P. ostreatus. Therefore, this study aims to explore the potential role of Po-milR-1 and Po-3KAT in P. ostreatus. By integrating the Po-milR-1 interference vector into the genome of P. ostreatus, it was found that the mycelial growth rate was accelerated and the fruiting body phenotype changed in the Po-STTM transformants. The expression analysis during development suggested an inverse expression pattern between Po-milR-1 and Po-3KAT. The analysis of qRT-PCR and HPLC showed that 2 mmol/L H₂O₂ promoted the expression of Po-3KAT, and the content of ergosterol increased to 1.17-fold, while 5 mmol/L H₂O₂ inhibited the expression of Po-3KAT, and the content of ergosterol decreased to 0.82-fold. In contrast to oxidative stress, 100 mmol/L and 250 mmol/L salt stress inhibited the expression of Po-3KAT, and the content of ergosterol decreased to 0.87-fold and 0.94-fold, respectively. These results suggest that Po-milR-1 and Po-3KAT may be involved in the regulation of growth, development, and stress responses in P. ostreatus, and may be associated with the change in ergosterol content. Full article

A coordinated extracellular matrix (ECM) and cytoskeletal remodeling are critical drivers of hepatocellular carcinoma (HCC) progression, yet the upstream post-transcriptional mechanisms regulating these processes in spontaneous canine HCC remain poorly defined. Previously, we reported significantly downregulated miRNAs and enriched pathways from differentially expressed genes (DEGs) in canine HCC tissues. In the present study, we investigated putative post-transcriptional target interactions between the prioritized miRNAs and enriched ECM–cytoskeleton pathway-associated genes (n = 34). Integrative bioinformatic analysis prioritized ACTG1 and SDC1 as the key putative targets of cfa-miR-10a based on their concordant prediction by four target-prediction algorithms, high transcript abundance, and inverse correlations with cfa-miR-10a-5p. RNAhybrid and miRanda analyses supported favorable predicted interactions between cfa-miR-10a and the 3′UTRs of both transcripts, with RNAhybrid minimum free energy values ≤ −20 kcal/mol and miRanda scores ≥ 140. In the matched canine transcriptomic subset, cfa-miR-10a and its predicted targets showed inverse expression trends. Cross-species analysis using TCGA-LIHC datasets further demonstrated concordant dysregulation of ACTG1, SDC1 and hsa-miR-10a-5p in human HCC, despite their weak or non-concordant correlation with hsa-miR-10a-5p. Collectively, these exploratory findings identify a candidate cfa-miR-10a–ACTG1/SDC1 network associated with coordinated ECM and cytoskeletal transcriptomic alterations in canine HCC. While cross-species analysis supports the conservation of target gene dysregulation, the divergent miRNA-mRNA correlation patterns highlight potential species-specific post-transcriptional co-expression landscapes that warrant future functional validation. Full article

Adult plant resistance (APR) is widely used in wheat breeding, but its behaviour across genetic backgrounds remains poorly understood. In this study, we analysed the expression of three APR loci (Lr34, Lr46, Lr67) following their introgression into elite winter wheat cultivars. BC₂F₁ populations derived from crosses between donor lines and commercial cultivars were evaluated under controlled infection with Puccinia triticina (Pt). Gene expression was assessed using RT-qPCR, and miRNA abundance was quantified by ddPCR at five time points (0–48 hours post-inoculation, hpi). Expression patterns differed markedly between genetic backgrounds, affecting both the magnitude and timing of gene activity. Lr34 and Lr67 showed the highest expression prior to inoculation, with no consistent or sustained induction following infection. In contrast, the candidate gene Lr46-Glu2 displayed a clear tendency towards early induction, with peak expression typically observed at 6–12 hours post-inoculation, although the amplitude of this response varied among genotypes. Levels of miRNA varied across genotypes and time points and did not consistently reflect mRNA expression. The results suggest that the expression of APR-associated loci is influenced by genetic background. Lr34 and Lr67 showed predominantly baseline-associated expression, whereas the Lr46-associated candidate gene Lr46-Glu2 displayed a more infection-responsive transcriptional profile following inoculation. These patterns are consistent with different transcriptional behaviours of the analysed genes but do not establish their functional contribution to resistance. Further phenotypic and functional validation will be required to determine the biological significance of these expression patterns. Full article

Background/Objectives: Loss of phosphatase and tensin homolog (PTEN) activity is a frequent feature of non-small-cell lung cancer (NSCLC), and epigenetic repression may contribute to its reduced expression. This study investigated the effects of 5-aza-2′-deoxycytidine (5-aza-dC) on PTEN, DNA methyltransferase 1 (DNMT1), PTEN promoter methylation-specific amplification patterns, and miR-148a-3p expression in NSCLC models. Methods: Publicly available cancer-genomics datasets were analyzed to compare PTEN and DNMT1 transcript abundance and to assess the association between PTEN methylation and transcript abundance in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). A549 and H460 cells were exposed to 2.5 or 5 µM 5-aza-dC for 72 h. Reverse-transcription quantitative PCR (RT-qPCR), Western blotting, methylation-specific PCR (MSP-PCR), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to evaluate RNA expression, protein abundance, methylation-specific amplification, and metabolic activity, respectively. Results: Bioinformatic analyses showed lower PTEN and higher DNMT1 expression in both NSCLC subtypes, together with inverse associations between PTEN methylation and transcript abundance. In both cell lines, 5-aza-dC reduced MTT metabolic activity and DNMT1 expression. PTEN mRNA and protein abundance increased significantly at 5 µM, whereas no significant changes were detected at 2.5 µM. MSP-PCR revealed persistent heterogeneous PTEN methylation-specific amplification patterns without clear evidence of progressive promoter demethylation. miR-148a-3p exhibited a biphasic response in A549 cells but remained unchanged in H460 cells. Conclusions: These findings support an association between 5-aza-dC exposure, increased PTEN expression, and reduced DNMT1 expression in NSCLC cells. Quantitative methylation analysis and mechanistic validation are required to clarify the contribution of miR-148a-3p to this regulatory association. Full article

PBU (N-phenyl-N′-thiazolylurea) promotes callus induction and adventitious bud differentiation in eucalyptus, but the miRNA-mediated regulatory mechanisms underlying these effects remain unclear. In this study, calli of Eucalyptus urophylla × E. grandis clone DH32-29 with distinct phenotypes were cultured at four PBU concentrations (0, 0.1, 1 and 5 mg L⁻¹) and analyzed by small-RNA sequencing, targeted metabolomics, and qRT-PCR validation. A total of 114 common differentially expressed miRNAs were identified, targeting 610 mRNAs. Functional enrichment analysis revealed that these targets were predominantly associated with lignin metabolism, phenylpropanoid metabolism, biotin metabolism, tryptophan metabolism, protein processing in the endoplasmic reticulum, and galactose metabolism. Metabolomic profiling detected 3029 metabolites, with differential metabolites enriched in the ABC transporter pathway, galloyl sugar biosynthesis, and cofactor biosynthesis. Key miRNA families, including miR164, miR165/166, and miR396, exhibited PBU concentration-dependent expression patterns and were predicted, based on in silico target prediction and qRT-PCR co-expression, to be potentially associated with target genes involved in lignin biosynthesis, ROS-related metabolism, and cytokinin homeostasis; these regulatory relationships remain to be experimentally validated. Among the tested concentrations, 1 mg L⁻¹ PBU was the dosage associated with the strongest reprogramming of secondary metabolism and with metabolic signatures suggestive of better preserved redox homeostasis; future work will build on this reference dataset with quantitative regeneration phenotyping and direct redox measurements to confirm this candidate optimum. These findings provide new insights into PBU-mediated in vitro regeneration in eucalyptus and offer a molecular basis for optimizing regeneration systems in E. urophylla × E. grandis. These findings provide new insights into the miRNA-metabolite regulatory network underlying phenylurea-mediated callus differentiation in woody plants. Full article