Search Results (877)

A dietary supplement, trans-resveratrol and hesperetin (tRES+HESP)—also known as GlucoRegulate—induces increased expression of glyoxalase 1 (Glo1) by activation of transcription factor Nrf2, countering accumulation of the reactive dicarbonyl glycating agent, methylglyoxal. tRES+HESP corrected insulin resistance and decreased fasting and postprandial plasma glucose and low-grade inflammation in overweight and obese subjects in a clinical trial. The aim of this study was to explore, for the first time, health-beneficial gene expression other than Glo1 induced by tRES+HESP in human endothelial cells and fibroblasts in primary culture and HepG2 hepatoma cell line and activity of cis-resveratrol (cRES) as a Glo1 inducer. We measured antioxidant response element-linked gene expression in these cells in response to 5 µM tRES+HESP by the NanoString method. tRES+HESP increases gene expression linked to the prevention of dicarbonyl stress, lipid peroxidation, oxidative stress, proteotoxicity and hyperglycemia-linked glycolytic overload. Downstream benefits were improved regulation of glucose and lipid metabolism and decreased inflammation, extracellular matrix remodeling and senescence markers. The median effective concentration of tRES was ninefold lower than cRES in the Glo1 inducer luciferase reporter assay. The GlucoRegulate supplement provides a new treatment option for the prevention of type 2 diabetes and metabolic dysfunction–associated steatotic liver disease and supports healthy aging. Full article

Background/Objectives: Different risk factors are involved in the initiation and progression of melanoma. In particular, genetic and epigenetic pathways are involved in all stages of melanoma and are exploited in therapeutic approaches. This study investigated the role of circular RNA circ_0001591 in melanoma cell migration. Methods: Three different melanoma cell lines were transfected with siRNA targeting circ_0001591 and with mimic or inhibitor molecules for miR-20a-3p and miR-34a-5p. Gene and protein expression levels were analyzed by RT-qPCR and Western blot, respectively. Dual luciferase reporter assays were performed to confirm the direct interaction of miR-20a-3p and miR-34a-5p with circ_0001591, as well as with the 3’UTRs of AXL (for both miRNAs) and FOSL1 (miR-34a-5p only). Wound healing assays were conducted to assess cell migration velocity. Results: The silencing of circ_0001591 significantly reduces the migration ability of melanoma cell lines. This downregulation was associated with an increased expression of miR-20a-3p and miR-34a-5p. Dual luciferase reporter assays confirmed the direct binding of both miRNAs to circ_0001591, supporting its role as a molecular sponge. The same assays also verified that miR-20a-3p directly targets the 3’UTR of AXL, while miR-34a-5p binds the 3’UTRs of both AXL and FOSL1. Western blot analysis showed that the modulation of this axis affects the expression levels of the AXL and FRA1 oncoproteins. Conclusions: Our findings demonstrate that circ_0001591 promotes melanoma migration by sponging miR-20a-3p and miR-34a-5p, thereby indirectly modulating the expression of AXL and FRA1 oncoprotein. Further investigations of this new regulatory network are needed to better understand its role in melanoma progression and to support the development of targeted therapies. Full article

Lonicera japonica Thunb. is a traditional Chinese medicinal herb whose floral buds are the primary source of pharmacological compounds that require manual harvesting. As a result, its floral bud duration, determined by the opening time, is a key determinant of both quality and economic value. However, the genetic mechanisms controlling floral bud duration remain poorly understood. In this study, we employed population structure analysis and molecular experiments to identify candidate genes associated with this trait. The improved cultivar Beihua No. 1 (BH1) opens its floral buds significantly later than the landrace Damaohua (DMH). Exogenous application of methyl jasmonate (MeJA) to BH1 indicated that jasmonate acts as a negative regulator of floral bud duration by accelerating floral bud opening. A genome-wide selection scan across 35 germplasms with varying floral bud durations identified the transcription factor LjWRKY50 as the causative gene influencing this trait. The dual-luciferase reporter assay and qRT-PCR experiments showed that LjWRKY50 activates the expression of the jasmonate biosynthesis gene, LjAOS. A functional variant within LjWRKY50 (Chr7:24636061) was further developed into a derived cleaved amplified polymorphic sequence (dCAPS) marker. These findings provide valuable insights into the jasmonate-mediated regulation of floral bud duration, offering genetic and marker resources for molecular breeding in L. japonica. Full article

Background: Preeclampsia (PE) is a pregnancy-specific disease and hypertensive disorder with a multifactorial pathogenesis involving complex molecular regulatory networks. Recent studies highlight the critical role of non-coding RNAs, particularly miRNAs and lncRNAs, in PE development. This study investigates the molecular interaction between miR-7151-5p and the lncRNA KCNQ1OT1 and their functional contributions to PE pathogenesis. Methods: An integrative approach combining RNAhybrid-based bioinformatics, dual-luciferase reporter assays, qRT-PCR, Transwell migration and invasion assays, and RNA sequencing was employed to characterize the binding between miR-7151-5p and KCNQ1OT1 and assess their influence on trophoblast cell function and gene expression. Results: A bioinformatic analysis predicted a stable binding site between miR-7151-5p and KCNQ1OT1 (minimum free energy: –37.3 kcal/mol). The dual-luciferase reporter assay demonstrated that miR-7151-5p directly targets KCNQ1OT1, leading to suppressed transcriptional activity. In HTR8/SVneo cells, miR-7151-5p overexpression significantly downregulated both KCNQ1OT1 and Notch1 mRNA, whereas its inhibition showed no significant changes, suggesting additional regulatory mechanisms of Notch1 expression. Transwell assays indicated that miR-7151-5p overexpression suppressed trophoblast cell migration and invasion, whereas its inhibition enhanced these cellular behaviors. RNA-seq analysis further revealed that miR-7151-5p overexpression altered key signaling pathways, notably the TGF-β pathway, and significantly modulates PE-associated genes, including PLAC1, ANGPTL6, HIRA, GLA, HSF1, and BAG6. Conclusions: The regulatory effect of miR-7151-5p on KCNQ1OT1, along with its influence on trophoblast cell dynamics via Notch1 and TGF-β signaling pathways, highlights its role in PE pathogenesis and supports its potential as a biomarker in early PE screening. Full article

Metformin is one of the most commonly used medications to treat type 2 diabetes. In addition to lowering blood sugar, it can also promote the regeneration of certain organs or tissues. Planarian Dugesia japonica, with its remarkable regenerative capacity, has become an important model organism for studying pharmacology and regenerative medicine. Planarian eyespot regeneration involves precise tissue regeneration via mechanisms like cell proliferation, differentiation, and gene regulation following body damage. Experiments on planarian eyespot regeneration have confirmed that 1 mM metformin significantly promotes regeneration. Through analysis of the regenerating planarian miRNA database and the metformin-treated transcriptome database, combined with target gene prediction by TargetScan, the DjmiR-27b/DjPax6 axis was finally determined as the research focus. qPCR showed that metformin significantly affects the expression levels of DjmiR-27b and DjPax6. DjPax6 was identified as the target gene of DjmiR-27b through dual luciferase reporter gene analysis. Functional experiments revealed that metformin regulates the expression of DjPax6 via DjmiR-27b, thereby influencing the regeneration of planarian eyespots. In situ hybridization showed that both DjmiR-27b and DjPax6 are expressed throughout the entire body. This study reveals the molecular mechanism of metformin regulating planarian regeneration through miRNA, providing further insights into its role in the field of regeneration. Full article

Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42–48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy. Full article

NF-Y transcriptional regulators play crucial roles in diverse biological processes in plants, primarily through the formation of NF-Y complexes that bind to specific DNA motifs. These complexes modulate the expression of downstream genes, which influence plant development and growth. In our research, the function of the NF-Y family C subunit member SlNF-YC9 gene in tomato was investigated with the CRISPR/Cas9 method. In contrast to the WT (wild type), the mutant CR-SlNF-YC9 exhibited a prominent protrusion at the fruit tip. The quantitative PCR analysis displayed that the transcription levels of genes associated with auxin transport (PIN4, PIN5, and PIN9) as well as auxin response genes (ARF7 and LAX3) were enhanced in the CR-SlNF-YC9 fruits than in the WT. Analysis of dual-luciferase reporter and EMSA assays showed that the SlNF-YC9-YB13b-YA7a trimer specifically binds the FUL2 promoter and represses its expression. In conclusion, our results suggest that SlNF-YC9 is crucial in influencing tomato fruit shape by the formation of NF-Y heterotrimeric complexes. Full article

The DNA mismatch repair (MMR) system plays a crucial role in repairing DNA damage and regulating cell cycle arrest induced by cadmium (Cd) stress. To elucidate the mechanism by which miRNAs target AtMSH2 in regulating Arabidopsis’ response to Cd stress, the wild-type Arabidopsis, Atmsh2 mutant, and three miRNA-overexpressing transgenic lines were grown hydroponically in half-strength MS solution containing cadmium (Cd) at concentrations of 0, 0.5, 1, 2, and 3 mg/L for 5 days. miRNA-seq analysis, bioinformatics prediction, dual-luciferase reporter assays, and qRT-PCR results demonstrated that miR5651, miR170-3p, and miR171a-3p specifically targeted AtMSH2 and their expression levels showed a significant negative correlation. Compared to wild-type (WT) Arabidopsis, Cd stress tolerance was significantly enhanced in miRNA-overexpressing transgenic lines. Moreover, exogenous application of these three miRNAs in half-strength MS liquid medium also markedly improved Cd stress tolerance in wild-type Arabidopsis. Furthermore, the expression of these three miRNAs expression was further upregulated by Cd stress in a dose-dependent manner. Additionally, DNA damage response in miRNA-overexpressing transgenic lines was promoted based on the expression of DNA repair, DNA damage signaling, and cell cycle genes, which differed from both wild-type and Atmsh2 plants. Taken together, miR5651, miR170-3p, and miR171a-3p participated in Cd stress response and improved plant Cd tolerance by mediating the expression of AtMSH2. Our study provides novel insights into the epigenetic mechanisms of Cd tolerance in plants, which sheds light on breeding for stress resilience in phytoremediation. Full article

Introduction: The interface between T cells and the tumor microenvironment, termed the ‘immunological synapse’, consists of multiple checkpoint protein pairs co-expressed on both sides of the synapse. mir-16, a microRNA from a widely known tumor-suppressor family of miRNAs, was previously shown by us to be downregulated in melanoma. As other miRNAs from this family have been shown to directly target checkpoint proteins, here we investigated whether miR-16 influences the expression patterns of checkpoint proteins in melanoma. Methods: Single-cell gene expression data from the melanoma microenvironment were retrieved from a public database. Melanoma cell lines were established from metastatic lesions and transiently transfected with an hsa-miR-16-5p-mimic RNA or a mir-16-expressing plasmid. The mRNA expression profiles were analyzed using an Affymetrix microarray. Direct targets of miR-16 were identified by luciferase reporter assays. Protein levels were assessed by Western blotting. Results: Bioinformatic analysis revealed that the expression levels of eight checkpoint mRNAs, known to be present on the melanoma side of the immunological synapse, were highly correlated. Four of these mRNAs contained putative binding sites for the miR-15/16 family. miR-16 expression was significantly reduced in melanoma cells, compared to normal melanocytes. Luciferase reporter assays demonstrated that miR-16 directly targets the 3′ untranslated regions (3′UTRs) of CD40, CD80. The mRNAs downregulated following miR-16 overexpression were highly enriched for genes involved in autophagy, vesicle-mediated transport, and the regulation of protein membrane localization. Among these, VTI1B and SMPD1 were confirmed to be direct targets of miR-16. Transient overexpression of miR-16 resulted in a significant reduction in SMPD1 and VTI1B levels in melanoma cell lines. Conclusions: Our findings suggest that miR-16 potentially modulates melanoma tumorigenesis, metastasis and immunogenicity by altering the composition of checkpoint proteins at the immunological synapse and by regulating cellular pathways associated with intracellular trafficking and transmembrane protein presentation. Full article

Background: Intramuscular fat content is positively correlated with meat flavor and juiciness. Increasing the intramuscular fat (IMF) content of chickens while increasing their growth rate has become a hot topic in molecular breeding. The group’s previous studies showed that miR-128-3p inhibited chicken intramuscular adipocyte differentiation and lipogenesis. However, the regulatory mechanism of miR-128-3p in intramuscular preadipocytes is currently unknown. In this study, we investigated the mechanism of miR-128-3p regulation of chicken intramuscular adipocyte differentiation and deposition. Results: Transcriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had seventeen significantly differentially expressed LincRNAs (p < 0.05), including six upregulated and eleven downregulated ones; the inhibitor-treated group had seventeen differentially expressed LincRNAs (p < 0.05), including eight upregulated and nine downregulated ones; and twenty-four differentially expressed LincRNAs (p < 0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with fourteen upregulated and ten downregulated ones. Functional enrichment analysis revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in the negative regulation of metabolic processes, response to steroid hormones, and regulation of actin cytoskeleton. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DELincRNAs, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2. LincRNA-MSTRG.673.2 was predominantly expressed in the cytoplasm of intramuscular adipocytes. Dual luciferase reporter identified the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2. The results of subsequent functional assays demonstrated that interfering with MSTRG.673.2 has been shown to inhibit lipid deposition in intramuscular preadipocytes. Transfection experiments have shown that LincRNA-MSTRG.673.2 can affect the expression of miR-128-3p. Conclusions: This study found that LincRNA-MSTRG.673.2 promoted chicken intramuscular adipocyte differentiation by downregulating miR-128-3p. The results are noteworthy for improving chicken meat quality, molecular breeding, and lipid metabolism research. Full article

The neurobiological mechanisms underlying methamphetamine use disorder (MUD) remain elusive, and specific treatment modalities as well as diagnostic markers are scarce. The emergence of exosomes has opened up possibilities for developing diagnostic and assessment biomarkers for neuropsychiatric disorders. Hence, the present study aimed to preliminarily explore the alterations in exosomal miRNA expression in MUD patients and the potential mechanisms involved in MUD. First, miRNA sequencing and RT-qPCR were used to verify the differential expression of peripheral blood exosomal miR-184-3p and miR-4433a-5p in MUD patients. Subsequently, the diagnostic ability of these two miRNAs for MUD was evaluated using ROC analysis. Finally, the regulatory relationship between miRNA-184-3p and its downstream target gene CRTC1 was verified by dual luciferase reporter assay. The results demonstrated that exosomal miR-184-3p and miR-4433a-5p were markedly decreased in MUD patients. However, the expression level of miR-4433a-5p was influenced by anxiety-depressive symptoms. The ROC analysis revealed that the AUCs of exosomal miRNA-184-3p in the training and validation sets of MUD patients were 0.902 and 0.823, respectively. In conclusion, exosomal miR-184-3p levels in peripheral blood may be a potential biomarker for the diagnosis and assessment of MUD, and it may be involved in the pathophysiological process of MUD through the targeted regulation of the CRTC1/CREB pathway. Full article

BEL1-like homeodomain protein 3 (BLH3) plays a crucial role in plant development. However, its involvement in the salt stress response has not been studied. In this study, we investigated the molecular mechanism underlying the response of LpBLH3 to salt stress in Lilium pumilum (L. pumilum) using various techniques, including quantitative PCR (RT-qPCR), determination of physiological indices of plant after Saline-Alkali stress, yeast two-hybrid screening, luciferase complementation imaging (LCI), and chromosome walking to obtain the promoter sequence, analyzed by PlantCARE, electrophoretic mobility shift assay (EMSA), and then dual-luciferase reporter assay(LUC). RT-qPCR analysis revealed that LpBLH3 is most highly expressed in the leaves of L. pumilum. The expression of LpBLH3 peaks at 24 or 36 h in the leaves under different saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling and stronger photosynthesis. The overexpression of LpBLH3 can enhance lignin accumulation in root and stem by positively modulating the expression of crucial genes within the lignin biosynthesis pathway. Y2H and LCI analyses demonstrated that LpBLH3 interacts with LpKNAT3. Additionally, EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 and upregulate the expression of ABI5 downstream genes (LpCAT1/LpATEM/LpRD29B). In summary, LpBLH3 enhances the plant’s salt tolerance through the ABA pathway and lignin synthesis. This study can enrich the functional network of the BLH transcription factor family, obtain Lilium pumilum lines with good saline-alkali resistance, expand the planting area of Lilium pumilum, and improve its medicinal and ornamental values. Additionally, the functional analysis of the BLH transcription factor family provides new insights into how crops adapt to the extreme growth environment of saline-alkali soils. Full article

Background/Objectives: The regulatory SNPs (rSNPs) that disturb the binding of transcription factors (TFs) and alter the transcription levels of genes play a paramount role in the formation of different traits and are associated with many pathologies. The search for allele-specific events in RNA-seq and ChIP-seq data is a powerful genome-wide approach to detect rSNPs. Using this approach, we have identified the T → A rs2072580 substitution in the bidirectional SART3/ISCU promoter as a potential rSNP and demonstrated its association with colorectal cancer, relying on International Cancer Genome Consortium data. The goal of this work was to identify the TF binding site that is affected by the T → A substitution and to study the effect of this substitution on reporter gene expression in different plasmid constructs. Methods: Electrophoretic mobility shift assay (EMSA), cross-competition analysis and supershift assay, plasmid construction, and dual luciferase reporter assay. Results: The T → A rs2072580 substitution is shown to damage the binding site for ubiquitous TF CREB1 and to significantly decrease the activity of the heterologous promoter carrying the cassettes of two or three repeated CREB binding sites inserted upstream of it. However, the substitution disturbing the CREB1 binding site within the bidirectional promoter shared by SART3 and ISCU inhibits the promoter activity of only the SART3 gene but has no effect on the activity of the ISCU promoter. Conclusions: The performed comprehensive functional analysis of the T → A rs2072580 in the bidirectional SART3/ISCU promoter unambiguously implies it is an rSNP. These results form the background for further studies of this rSNP and its potential significance for various pathologies. Full article

Strawberry (Fragaria × ananassa Duch.) is an important fruit worldwide whose growth, development, and productivity are threatened by salinity. The WRKY transcription factors (TFs) were reported to play an important role in regulating abiotic stresses response. However, research on their roles to regulate salt stress tolerance in strawberry remains limited. In current study, the FvWRKY75 gene was isolated and characterized from the Ruegen strawberry, and induced by various stress treatment. The results showed that the FvWRKY75 transcription factor was a transcriptional activator and localized in the nucleus. Phenotypic and physiological analysis revealed that ectopic expression of FvWRKY75 in Arabidopsis improved salt tolerance by enhancing the antioxidant system activities, modulating ROS scavenging and upregulating stress-related genes. Y1H and dual luciferase assays revealed that FvWRKY75 can directly bind to the promoter of the FvCRK5 gene by recognizing the W-box element. Compared with the WT, ectopic expression of FvCRK5 gene in Arabidopsis enhanced salt tolerance characterized by the reduced ROS accumulation, higher chlorophyll content, lower MDA content, and enhanced SOD and POD activity. Herein, the FvWRKY75 gene acted as a positive regulator in salt stress resistance, at least in part, via the WRKY-CRK network to regulate the antioxidant enzyme defense system and stress-related genes to regulate salt stress tolerance in strawberry. Full article

Anthocyanins, as natural pigments belonging to the flavonoid group, play a crucial role in plant reproduction, stress resistance and human fitness. Kiwifruit, which is rich in anthocyanins, demonstrates significant potential for promoting health benefits. Although light is widely recognized as an inducer of anthocyanin accumulation, we observed that kiwifruit accumulates more anthocyanin after bagging treatment. This unexpected finding suggests that anthocyanin biosynthesis in kiwifruit may also be regulated by other environmental or physiological factors influenced by bagging, such as humidity, temperature, or gas exchange. This implies that bagging may trigger specific regulatory pathways that promote anthocyanin accumulation through multiple environmental cues beyond light. Therefore, RNA-seq was performed to find the potential pathway. A total of 260 differentially expressed genes were found, including 8 transcription factors and 1 anthocyanin biosynthesis gene F3GT1 (glucosyltransferase). Dual-luciferase reporter assays revealed that bHLH transcription factors could activate the promoter of F3GT1 by 2.45-fold. We infer that bagging treatment increases the kiwifruit anthocyanin content through the bHLH291-F3GT1 pathway. This study not only highlights the potential agricultural applications and commercial value of bagging treatment but also provides new theoretical support for improving fruit coloration and optimizing breeding strategies. Full article