Regulation Of Downstream Genes Research Articles

Pseudorabies virus inhibits the unfolded protein response for viral replication during the late stages of infection.

Pseudorabies virus (PRV) poses a significant threat to the global swine breeding industry and public health, but how the virus transverses the host defense systems for efficient viral replication and pathogenesis remains unclear. Here, we report that PRV could inhibit the unfolded protein response (UPR), a critical component of host innate immunity against viral infection, to promote virus replication during the late infection stages. PERK was shown phosphorylated and active in PRV-infected cells, but the subsequent events were suppressed post virus infection, such as eIF2α phosphorylation, ATF4 expression, and the formation of stress granules (SGs). In the meantime, although IRE1α was also active, its activated effector XBP1s was suppressed through downregulation of XBP1 mRNA levels and cleavage of XBP1s protein. Our findings also indicate that the Golgi apparatus, where ATF6 activation occur, was severely damaged in PRV-infected cells. Meanwhile, the downstream regulatory genes associated with the three UPR sensors, such as ERp60, CHOP, and EDEM1, remained silent in PRV-infected cells. Enhanced viral replication was observed post knockdown of UPR effectors ATF4 or XBP1, while stimulation with UPR activators inhibits virus replication. In conclusion, our findings address the critical question of how PRV regulates cellular UPR in favor of viral replication, and expand understanding of viruses mediated UPR suppression in general.

P0104 Decoding cytokine networks in ulcerative colitis to identify pathogenic mechanisms and therapeutic targets

Abstract Background Cytokines are key signalling molecules of the immune system that play critical roles in immune regulation. Cytokine ligands binding to their receptors initialise signalling cascades, culminating in the regulation of downstream target genes, including other cytokines. Thus, cytokines can form complex networks of interactions, in which cytokines regulate the production and activity of other cytokines, thereby organising immune responses. Such cytokine–cytokine interactions are important for maintaining the proper balance and specificity of immune responses. However, when dysregulated, chronic immune-mediated inflammatory disorders such as ulcerative colitis (UC) can develop. Despite the advent of advanced therapies targeting cytokine signalling, treatment outcomes for UC patients remain suboptimal. As such, there is a pressing need to better understand the cytokine regulation in UC by comprehensively mapping the interconnected cytokine signalling networks perturbed in UC patients. Methods We undertook single-cell systems immunology modelling of colonic biopsies of treatment-naive and treatment-exposed UC patients and healthy controls using harmonised single-cell transcriptomics data from the scIBD atlas [1]. This dataset allowed us to build condition specific cytokine signalling networks underpinned by putative cytokine–cytokine interactions. Results The generated cytokine networks effectively captured known physiologically relevant cytokine–cytokine interactions, which we recapitulated in vitro using UC patient-derived colonic epithelial organoids (n = 4). The resulting cytokine networks revealed new aspects of UC pathogenesis, including a cytokine subnetwork unique to treatment-naive UC patients. We identified highly rewired cytokines across UC disease states (TL1A, IL22, IL23A, and OSM), and JAK paralogue-specific cytokine–cytokine interactions that may inform the broader cytokine network effects of JAK inhibitor treatment. The cytokine network captures all significantly downregulated cytokine targets of anti-TL1A treatment shown in Hassan-Zahraee et al. [2], highlighting TL1A as an important upstream regulator of TNF and IL23A. Conclusion These findings open up novel avenues for guiding future cytokine-targeting therapeutic approaches in UC, by highlighting the likely downstream cytokines perturbed by cytokine inhibition, and elucidating the cytokine signalling differences in treatment-naive and treatment exposed UC. The presented methodology [3] can be readily applied to gain similar insights into other immune-mediated inflammatory diseases (IMIDs).

The Role of NDRG2 in the Chronic Pain Process

Chronic pain is an important physiological response of the organism that involves a variety of molecules and cells, among which astrocytes play an important role in the development of chronic pain. NDRG2, a member of the N-myc downstream regulatory gene family, is expressed in astrocytes of the central nervous system and is involved in physiological functions affecting apoptosis, astrocyte activation, blood-brain barrier integrity and glutamate clearance regulation of glutamate clearance and other physiological functions. In this paper, we focus on the involvement of NDRG2 in the regulation of chronic pain by affecting the function of astrocytes, and discuss whether NDRG2 can be used as a potential target for pain treatment, taking into account the existing studies.

The SnRK2.2-ZmHsf28-JAZ14/17 module regulates drought tolerance in maize.

Abscisic acid (ABA) and jasmonic acid (JA) are important plant hormones in response to drought stress. We have identified that ZmHsf28 elevated ABA and JA accumulation to confer drought tolerance in maize; however, the underlying mechanism still remains elusive. The knockout line zmhsf28 is generated to confirm the positive role of ZmHsf28 in drought response. Multiple approaches are combined to reveal protein interaction among ZmHsf28, ZmSnRK2.2 and ZmJAZ14/17, which form a regulatory module to mediate maize drought tolerance through regulating ABA and JA key biosynthetic genes ZmNCED3 and ZmLOX8. Upon drought stress, zmhsf28 plants exhibit weaker tolerance than the WT plants with slower stomatal closure and more reactive oxygen species accumulation. ZmHsf28 interacted with ZmSnRK2.2 physically, resulting in phosphorylation at Ser220, which enhances binding to the heat shock elements of ZmNECD3 and ZmLOX8 promoters and subsequent gene expression. Meanwhile, ZmMYC2 upregulates ZmHsf28 gene expression through acting on the G-box of its promoter. Besides, ZmJAZ14/17 competitively interact with ZmHsf28 to interfere with protein interaction between ZmHsf28 and ZmSnRK2.2, blocking ZmHsf28 phosphorylation and impairing downstream gene regulation. The ZmSnRK2.2-ZmHsf28-ZmJAZ14/17 module is identified to regulate drought tolerance through coordinating ABA and JA signaling, providing the insights for breeding to improve drought resistance in maize.

An organotypic atlas of human vascular cells

The human vascular system, comprising endothelial cells (ECs) and mural cells, covers a vast surface area in the body, providing a critical interface between blood and tissue environments. Functional differences exist across specific vascular beds, but their molecular determinants across tissues remain largely unknown. In this study, we integrated single-cell transcriptomics data from 19 human organs and tissues and defined 42 vascular cell states from approximately 67,000 cells (62 donors), including angiotypic transitional signatures along the arterial endothelial axis from large to small caliber vessels. We also characterized organotypic populations, including splenic littoral and blood–brain barrier ECs, thus clarifying the molecular profiles of these important cell states. Interrogating endothelial–mural cell molecular crosstalk revealed angiotypic and organotypic communication pathways related to Notch, Wnt, retinoic acid, prostaglandin and cell adhesion signaling. Transcription factor network analysis revealed differential regulation of downstream target genes in tissue-specific modules, such as those of FOXF1 across multiple lung vascular subpopulations. Additionally, we make mechanistic inferences of vascular drug targets within different vascular beds. This open-access resource enhances our understanding of angiodiversity and organotypic molecular signatures in human vascular cells, and has therapeutic implications for vascular diseases across tissues.

The PHD transcription factor ThPHD5 regulates antioxidant enzyme activity to increase salt tolerance in Tamarix hispida

PHD proteins are an important class of transcription factors (TFs) that are widely distributed in eukaryotes and play crucial roles in many aspects of plant growth, development and response to stress. We identified a transcription factor, ThPHD5, from the PHD family in Tamarix hispida based on its potential involvement in abiotic stress response processes. In this study, the salt tolerance function of ThPHD5 from T. hispida was further characterized. The qRT-PCR results showed ThPHD5 expression was significantly induced by NaCl, PEG and ABA treatments. Transient transformation analysis revealed that ThPHD5 improved salt tolerance in T. hispida by increasing POD and SOD activity, decreasing the MDA, total ROS content and electrolyte leakage. To explore the salt tolerance mechanism of the ThPHD5 TF, its binding DNA motifs and potential downstream regulatory genes were analyzed. The results showed that ThPHD5 affect the expression of 7 antioxidant enzyme-related genes. The Yeast one-hybrid (Y1H) and Electrophoretic Mobility Shift Assay (EMSA) results indicated ThPHD5 could bind to ABRE, MYB and Dof cis-acting elements. ChIP-PCR further confirmed ThPHD5 exercise its regulatory function by directly binding motifs on the ThPOD16, ThSOD and ThSOD1 promoters. Taken together, these findings indicate the ThPHD5 TF improves salt tolerance in T. hispida by regulating the expression of antioxidant enzyme-related genes to increase antioxidant enzyme activity, enhance the ROS scavenge ability, reduce ROS accumulation and cellular damage.

Systems Biology Methods via Genome-Wide RNA Sequences to Investigate Pathogenic Mechanisms for Identifying Biomarkers and Constructing a DNN-Based Drug–Target Interaction Model to Predict Potential Molecular Drugs for Treating Atopic Dermatitis

This study aimed to construct genome-wide genetic and epigenetic networks (GWGENs) of atopic dermatitis (AD) and healthy controls through systems biology methods based on genome-wide microarray data. Subsequently, the core GWGENs of AD and healthy controls were extracted from their real GWGENs by the principal network projection (PNP) method for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Then, we identified the abnormal signaling pathways by comparing the core signaling pathways of AD and healthy controls to investigate the pathogenesis of AD. Then, IL-1β, GATA3, Akt, and NF-κB were selected as biomarkers for their important roles in the abnormal regulation of downstream genes, leading to cellular dysfunctions in AD patients. Next, a deep neural network (DNN)-based drug–target interaction (DTI) model was pre-trained on DTI databases to predict molecular drugs that interact with these biomarkers. Finally, we screened the candidate molecular drugs based on drug toxicity, sensitivity, and regulatory ability as drug design specifications to select potential molecular drugs for these biomarkers to treat AD, including metformin, allantoin, and U-0126, which have shown potential for therapeutic treatment by regulating abnormal immune responses and restoring the pathogenic signaling pathways of AD.

IL23R mutations associated with decreased risk of psoriasis lead to the differential expression of genes implicated in the disease.

Psoriasis is an incurable immune-mediated skin disease, affecting around 1%-3% of the population. Various lines of evidence implicate IL23 as being pivotal in disease. Genetic variants within the IL23 receptor (IL23R) increase the risk of developing psoriasis, and biologic therapies specifically targeting IL23 demonstrated high efficacy in treating disease. IL23 acts via the IL23R, signalling through the STAT3 pathway, mediating the cascade of events that ultimately results in the clinical presentation of psoriasis. Given the essential role of IL23R in disease, it is important to understand the impact of genetic variants on receptor function with respect to downstream gene regulation. Here we developed model systems in CD4+ (Jurkat) and CD8+ (MyLa) T cells to express either the wild type risk or mutant (R381Q) protective form of IL23R. After confirmation that the model system expressed the genes/proteins and had a differential effect on the phosphorylation of STAT3, we used RNAseq to explore differential gene regulation, in particular for genes implicated with risk to psoriasis, at a single time point for both cell types, and in a time course experiment for Jurkat CD4+ T cells. These experiments discovered differentially regulated genes in the cells expressing wild type and mutant IL23R, including HLA-B, SOCS1, RUNX3, CCR5, CXCR3, CCR9, KLF3, CD28, IRF, SOCS6, TNFAIP and ICAM5, that have been implicated in both the IL23 pathway and psoriasis. These genes have the potential to define a IL23/psoriasis pathway in disease, advancing our understanding of the biology behind the disease.

H3K9 lactylation in malignant cells facilitates CD8+ T cell dysfunction and poor immunotherapy response

Histone lysine lactylation (Kla) is a post-translational modification, and its role in tumor immune escape remains unclear. Here, we find that increased histone lactylation is associated with poor response to immunotherapy in head and neck squamous cell carcinoma (HNSCC). H3K9la is identified as a specific modification site in HNSCC. Using cleavage under targets and tagmentation analyses, interleukin-11 (IL-11) is identified as a downstream regulatory gene of H3K9la. IL-11 transcriptionally activates immune checkpoint genes through JAK2/STAT3 signaling in CD8+ Tcells. Additionally, IL-11 overexpression promotes tumor progression and CD8+ Tcell dysfunction invivo. Moreover, IL11 knockdown reverses lactate-induced CD8+ Tcell exhaustion, and cholesterol-modified siIL11 restores CD8+ Tcell killing activity and enhances immunotherapy efficacy. Clinically, H3K9la positively correlates with IL-11 expression and unfavorable immunotherapy responses in patients. This study reveals the crucial role of histone lactylation in immune escape, providing insights into immunotherapy strategies for HNSCC.

Regulation of Cell Membrane Potential through Supramolecular System for Activating Calcium Ion Channels.

The regulation of the cell membrane potential plays a crucial role in governing the transmembrane transport of various ions and cellular life processes. However, in situ and on-demand modulation of cell membrane potential for ion channel regulation is challenging. Herein, we have constructed a supramolecular assembly system based on water-soluble cationic oligo(phenylenevinylene) (OPV) and cucurbit[7]uril (CB[7]). The controllable disassembly of OPV/4CB[7] combined with the subsequent click reaction provides a step-by-step adjustable surface positive potential. These processes can be employed in situ on the plasma membrane to modulate the membrane potential on-demand for precisely controlling the activation of the transient receptor potential vanilloid 1 (TRPV1) ion channel and up-regulating exogenous calcium-responsive gene expression. Compared with typical optogenetics, electrogenetics, and mechanogenetics, our strategy provides a perspective supramolecular genetics toolbox for the regulation of membrane potential and downstream intracellular gene regulation events.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara)

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

RUNX1 isoforms regulate RUNX1 and target genes differentially in platelets-megakaryocytes: association with clinical cardiovascular events

BackgroundHematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoters to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream gene regulation in megakaryocytes and platelets are unknown. ObjectivesTo understand the regulation of RUNX1 and its target genes by RUNX1 isoforms. MethodsWe performed studies on RUNX1 isoforms in megakaryocytic human erythroleukemia (HEL) cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD). ResultsIn chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells, RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes (MYL9, F13A1, PCTP, PDE5A, and others) differentially in HEL cells. In platelets, RUNX1B transcripts (by RNA sequencing) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with F13A1, PCTP, PDE5A, RAB1B, and others, and negatively with MYL9. In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets F13A1 and RAB31 associated with acute events. ConclusionRUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner, and this is associated with acute events in CVD.

Genome-Wide Transcriptome Analysis of a Virulent sRNA, Trans217, in Xanthomonas oryzae pv. oryzae (Xoo), the Causative Agent of Rice Bacterial Blight.

Small non-coding RNAs (sRNAs) act as post-transcriptional regulators to participate in many cellular processes. Among these, sRNA trans217 has been identified as a key virulent factor associated with pathogenicity in rice, triggering hypersensitive reactions in non-host tobacco and facilitating the secretion of the PthXo1 effector in Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A. Elucidating potential targets and downstream regulatory genes is crucial for understanding cellular networks governing pathogenicity and plant resistance. To explore the targets regulated by sRNA trans217, transcriptome sequencing was carried out to assess differential expression genes (DEGs) between the wild-type strain PXO99A and a mutant lacking the sRNA fragment under both virulence-inducing or normal growth conditions. DEG analysis revealed that sRNA trans217 was responsible for diverse functions, such as type III secretion system (T3SS), glutamate synthase activity, and oxidative stress response. Three genes were selected for further investigation due to their significant differential expression and biological relevance. Deletion of PXO_RS08490 attenuated the pathogenicity of Xoo in rice and reduced the tolerance level of PXO99A to hydrogen peroxide. These findings suggest a regulatory role of sRNA trans217 in modulating bacterial virulence through multiple gene targets, either directly or indirectly.

Allele-Specific Hormone Dynamics in Highly Transgressive F2 Biomass Segregants in Sugarcane (Saccharum spp.).

Sugarcane holds global promise as a biofuel feedstock, necessitating a deep understanding of factors that influence biomass yield. This study unravels the intricate dynamics of plant hormones that govern growth and development in sugarcane. Transcriptome analysis of F2 introgression hybrids, derived from the cross of Saccharum officinarum "LA Purple" and wild Saccharum robustum "MOL5829", was conducted, utilizing the recently sequenced allele-specific genome of "LA Purple" as a reference. A total of 8059 differentially expressed genes were categorized into gene models (21.5%), alleles (68%), paralogs (10%), and tandemly duplicated genes (0.14%). KEGG analysis highlighted enrichment in auxin (IAA), jasmonic acid (JA), and abscisic acid (ABA) pathways, revealing regulatory roles of hormone repressor gene families (Aux/IAA, PP2C, and JAZ). Signaling pathways indicated that downregulation of AUX/IAA and PP2C and upregulation of JAZ repressor genes in high biomass segregants act as key players in influencing downstream growth regulatory genes. Endogenous hormone levels revealed higher concentrations of IAA and ABA in high biomass, which contrasted with lower levels of JA. Weighted co-expression network analysis demonstrated strong connectivity between hormone-related key genes and cell wall structural genes in high biomass genotypes. Expression analysis confirmed the upregulation of genes involved in the synthesis of structural carbohydrates and the downregulation of inflorescence and senescence-related genes in high biomass, which suggested an extended vegetative growth phase. The study underscores the importance of cumulative gene expression, including gene models, dominant alleles, paralogs, and tandemly duplicated genes and activators and repressors of disparate hormone (IAA, JA, and ABA) signaling pathways are the points of hormone crosstalk in contrasting biomass F2 segregants and could be applied for engineering high biomass acquiring varieties.

Insight into the therapeutic effects of artesunate in relieving metabolic-associated steatohepatitis from transcriptomic and lipidomics analyses.

Artesunate (ART) is a water-soluble derivative of artemisinin, which has shown anti-inflammatory, anti-tumor, and immunomodulating effects. We aimed to investigate the potential therapeutic effects and mechanisms of ART in metabolic dysfunction-associated steatohepatitis (MASH). The mice were randomly divided into the control group, high-fat, high-cholesterol diet-induced MASH group, and the MASH treated with ART (30 mg/kg once daily) group. Liver enzymes, lipids, and histological features were compared among groups. The molecular mechanisms were studied by transcriptomic and lipidomics analyses of liver tissues. The mice of the MASH group had significantly increased hepatic fat deposition and inflammation in terms of biochemical indicators and pathological manifestations than the control group. The ART-treated group had improved plasma liver enzymes and hepatic cholesterol, especially at week 4 of intervention (p < 0.05). A total of 513 differentially expressed genesand 59 differentially expressed lipids were identified in the MASH group and the MASH+ART group. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment test showed that ART regulated glycerolipid metabolism pathway and enhanced fatty acid degradation. Peroxisome proliferator-activated receptor (PPAR)-α acted as a key transcription factor in the treatment of MASH with ART, which was confirmed by cell experiment. ART significantly improved fat deposition and inflammatory manifestations in MASH mice, with potential therapeutic effects. The mechanism of artemisinin treatment for MASH may involve extensive regulation of downstream genes by upstream transcription factors, such as PPAR-α, to restore hepatic lipid homeostasis.

RNA-Independent Regulatory Functions of lncRNA in Complex Disease.

During the metagenomics era, high-throughput sequencing efforts both in mice and humans indicate that non-coding RNAs (ncRNAs) constitute a significant fraction of the transcribed genome. During the past decades, the regulatory role of these non-coding transcripts along with their interactions with other molecules have been extensively characterized. However, the study of long non-coding RNAs (lncRNAs), an ncRNA regulatory class with transcript lengths that exceed 200 nucleotides, revealed that certain non-coding transcripts are transcriptional "by-products", while their loci exert their downstream regulatory functions through RNA-independent mechanisms. Such mechanisms include, but are not limited to, chromatin interactions and complex promoter-enhancer competition schemes that involve the underlying ncRNA locus with or without its nascent transcription, mediating significant or even exclusive roles in the regulation of downstream target genes in mammals. Interestingly, such RNA-independent mechanisms often drive pathological manifestations, including oncogenesis. In this review, we summarize selective examples of lncRNAs that regulate target genes independently of their produced transcripts.

MiR-338–5p, a novel metastasis-related miRNA, inhibits triple-negative breast cancer progression by targeting the ETS1/NOTCH1 axis

Breast cancer ranks as the most prevalent cancer globally, surpassing lung cancer, with recurrence/metastasis to be its main account for the cancer-related mortality. MicroRNAs (miRNAs) participate critically in various physiological and pathological processes through posttranscriptional regulation of downstream genes. Our preliminary findings identified miR-338–5p, potentially linked to metastasis in breast cancer, a previously unexplored area. Analysis of the GSE38867 dataset revealed the decreased miR-338–5p expression in metastatic breast cancer compared to normal tissues. Cellular function experiments and a xenograft tumor model demonstrated the inhibitory function of miR-338–5p on the progression of breast cancer in vitro and in vivo. Furthermore, it downregulated the expression of mesenchymal biomarkers and NOTCH1 significantly. With the predicting targets of miR-338–5p and transcription factors of the NOTCH1 gene, coupled with dual luciferase reporter assays, it is identified ETS1 as the interactor between miR-338–5p and NOTCH1. In breast cancer tissues, as well as in our xenograft tumor model, expression of ETS1 and NOTCH1 was positively correlated using immunohistochemical staining. This study reports, for the first time, on the miR-338–5p/ETS1/NOTCH1 axis and its pivotal role in breast cancer proliferation and metastasis. These findings propose a novel therapeutic strategy for breast cancer patients and lays a foundation for its clinical detection and treatment evaluation.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets-Megakaryocytes: Association with Clinical Cardiovascular Events.

Hematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoter to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream-gene regulation in megakaryocytes and platelets are unknown. To understand the regulation of RUNX1 and its target genes by RUNX1 isoforms. We performed studies on RUNX1 isoforms in megakaryocytic HEL cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD). In chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes (MYL9, F13A1, PCTP, PDE5A and others) differentially in HEL cells. In platelets RUNX1B transcripts (by RNAseq) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with F13A1, PCTP, PDE5A, RAB1B, and others, and negatively with MYL9. In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets F13A1 and RAB31 associated with acute events. RUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner and this associates with acute events in CVD.

Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21–3p, along with the downregulation of hsa-miR-152–3p and hsa-miR-186–5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

#1110 The AHR antagonist CH223191 alleviates kidney injury and renal fibrosis in adenine-induced chronic kidney disease

Abstract Background and Aims The aryl hydrocarbon receptor (AhR) is a transcription factor that can be activated by a variety of endogenous and exogenous ligands, and is involved in varieties of biological processes. It has been confirmed that the expression of AhR in kidney is elevated in chronic kidney disease (CKD). However, the specific role of AhR signaling pathway in CKD and its molecular mechanism remain unclear. This study aims to investigate the role and underlying mechanism of AhR in CKD, providing potential targets for the treatment of CKD. Method Male C57/B6L mice were randomly divided into control group, model group and treatment group. The model group was fed 0.2% adenine diet for 14 days to establish CKD model, while the treatment group was treated with AhR antagonist CH223191. AhR agonist kynurenine and antagonist CH223191 was applied in mouse tubular epithelial (TCMK-1) cells to investigate the underlying mechanisms of AhR in vitro. The renal function, histological changes and renal fibrosis in mice were evaluated to explore the role of AhR in adenine-induced CKD. Histological changes were evaluated by PAS and MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. Results The levels of serum creatinine (p &amp;lt; 0.0001) and urea nitrogen (p &amp;lt; 0.0001) in the model group were significantly increased, with obvious renal tubule dilation and renal fibrosis by PAS and MASSON staining. CH223191 treatment significantly reduced serum creatinine (p=0.0012) and urea nitrogen ( &amp;lt; 0.0001) levels in mice, and alleviated renal fibrosis histologically. Q-PCR and Western-Blot results showed that the renal expression level of AhR in the treatment group was decreased, while the expression levels of fibronectin (Fn) (p &amp;lt; 0.0001), α-smooth muscle actin (α-SMA) (p &amp;lt; 0.0001), type I collagen (p=0.0007) and transforming growth factor-β (TGF-b) (p=0.0027) were significantly decreased. In addition, the expression levels of N-GAL (p &amp;lt; 0.0001) and KIM-1 ( &amp;lt; 0.0001) molecular markers associated with kidney injury were reduced, suggesting that inhibiting AhR expression in vivo can reduce adenine-mediated kidney injury and renal fibrosis in CKD. In vitro experiments indicated that kynurenic acid could induce the expression of AhR and AhR downstream regulatory gene CYP1b1 and upregulated fibrosis-related genes in tubular epithelial cells, while CH223191 treatment downregulated the fibrosis-related genes in tubular epithelial cells. Conclusion AhR mediates renal injury and renal fibrosis in adenine-induced CKD mice. AhR antagonist CH223191 exerts a renal protective effect by reducing the expression of AhR in renal tubular epithelial cells, thereby alleviating renal injury and renal fibrosis.