Expression Levels Of Target Genes Research Articles

PCSK9i promoting the transformation of AS plaques into a stable plaque by targeting the miR-186-5p/Wipf2 and miR-375-3p/Pdk1/Yap1 in ApoE-/- mice.

Atherosclerosis (AS) is a multifaceted disease characterized by disruptions in lipid metabolism, vascular inflammation, and the involvement of diverse cellular constituents. Recent investigations have progressively underscored the role of microRNA (miR) dysregulation in cardiovascular diseases, notably AS. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) can effectively reduce circulating levels of low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp (a)], potentially fostering a more enduring phenotype for AS plaques. However, the underlying mechanisms by which PCSK9i enhances plaque stability remain unclear. In this study, we used microarray and bioinformatics techniques to analyze the regulatory impacts on gene expression pertinent to AS, thereby unveiling potential mechanisms underlying the plaque-stabilizing attributes of PCSK9i. ApoE-/- mice were randomly allocated into control, AS, PCSK9i, and Atorvastatin groups. The AS model was induced through a high-fat diet (HFD), succeeded by interventions: the PCSK9i group was subjected to subcutaneous SBC-115076 injections (8 mg/kg, twice weekly), and the Atorvastatin group received daily oral Atorvastatin (10 mg/kg) while on the HFD. Subsequent to the intervention phase, serum analysis, histological assessment using hematoxylin and eosin (H&E) and Oil Red O staining, microarray-centered miRNA analysis utilizing predictions from TargetScan and miRTarBase, and analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed to illuminate potential pathways. Real-time fluorescence quantitative PCR (RT-qPCR) was employed to quantify the expression levels of target genes. In comparison to the control group, the AS group displayed a significant elevation in blood lipid levels. Both PCSK9i and Atorvastatin effectively attenuated blood lipid levels, with PCSK9i exhibiting a more pronounced lipid-lowering impact, particularly concerning TG and LDL-C levels. Over the course of AS progression, the expression levels of mmu-miR-134, mmu-miR-141-5p, mmu-miR-17-3p, mmu-miR-195-3p, mmu-miR-210, mmu-miR-33-5p, mmu-miR-410, mmu-miR-411-5p, mmu-miR-499, mmu-miR-672-5p, mmu-miR-675-3p, and mmu-miR-301b underwent dynamic fluctuations. PCSK9i significantly down-regulated the expression of mmu-miR-186-5p, mmu-miR-222, mmu-miR-375-3p, and mmu-miR-494-3p. Further enrichment analysis disclosed that mmu-miR-186-5p, mmu-miR-222, mmu-miR-375-3p, and mmu-miR-494-3p were functionally enriched for cardiovascular smooth muscle cell proliferation, migration, and regulation. RT-qPCR results manifested that, in comparison to the AS group, PCSK9i significantly upregulated the expression of Wipf2, Pdk1, and Yap1 (p < 0.05). Aberrant miRNA expression may play a pivotal role in AS progression in murine models of AS. The subcutaneous administration of PCSK9i exerted anti-atherosclerotic effects by targeting the miR-186-5p/Wipf2 and miR-375-3p/Pdk1/Yap1 axes, thereby promoting the transition of AS plaques into a more stable form.

Development of a necroptosis-related prognostic model for uterine corpus endometrial carcinoma

Necroptosis is a recently identified caspase-independent form of cell death which plays a significant role in the onset and progression of cancer. MicroRNAs (miRNAs) are vital for the development of uterine corpus endometrial carcinoma (UCEC) because they are an important regulatory component in necroptosis. This study developed a new necroptosis-related miRNAs profile to predict the prognosis of patients with UCEC. The TCGA-UCEC cohort’s RNA sequencing data, consisting of 534 tumor samples and 33 normal samples, was downloaded. Ten differentially expressed miRNAs related to necroptosis were identified. A prediction model for necroptosis-related miRNAs was then created through COX regression and nomograms analysis. Clinical and pathological parameters were integrated to construct a nomogram and evaluate the model. Prognosis-related miRNAs were further used to predict target genes, and functional analysis was conducted to explore the potential mechanisms of these target genes. Subsequently, immune infiltration analysis was performed using transcriptome data to identify immune genes associated with prognosis, and the expression levels of target gene was validated using UCEC tissues. We identified 7 up-regulated miRNAs (hsa-miR-577, hsa-miR-7-5p, hsa-miR-210-3p, hsa-miR-210-5p, hsa-miR-200a-5p, hsa-miR-141-3p, hsa-miR-425-5p) and 3 down-regulated miRNAs (hsa-miR-7-2-3p, hsa-miR-383-5p, hsa-miR-29a-3p). The risk signature was based on univariate and multivariate COX analyses, constructed using 2 independent prognostic factors and miRNAs (hsa-miR-425-5p, hsa-miR-7-5p) associated with necroptosis. Nomograms demonstrated the prognostic value of risk level, age, FIGO stage, and histological type. Kaplan–Meier analysis revealed significant differences in overall survival (OS) outcomes associated with the expression of hsa-miR-425-5p (P < 0.001) and hsa-miR-7-5p (P = 0.015). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) investigations indicated that these miRNAs play crucial roles in tumor development, metastasis, and prognosis. Immune infiltration analysis showed decreased infiltration of CD8+ T cells, CD8+ T cells, NK cells, and M1 macrophages in normal tissues. Subsequently, a necroptosis-related immune gene significantly associated with prognosis (THRB) was identified, western blot and immunohistochemical staining confirmed the differential expression of THRB in normal endometrial tissues and tumor. Our findings demonstrate a close association between necroptosis and UCEC. The two necroptosis-related miRNAs used in this study may serve as valuable prognostic markers for UCEC patients, and are associated with immune cell infiltration. This suggests that necroptosis may be involved in the development of UCEC through its interaction with immune responses.

Identification of anti-gastric cancer effects and molecular mechanisms of resveratrol: From network pharmacology and bioinformatics to experimental validation.

Gastric cancer (GC) is one of the most aggressive malignancies with limited therapeutic options and a poor prognosis. Resveratrol, a non-flavonoid polyphenolic compound found in a variety of Chinese medicinal materials, has shown excellent anti-GC effect. However, its exact mechanisms of action in GC have not been clarified. To identify the effects of resveratrol on GC progression and explore the related molecular mechanisms. Action targets of resveratrol and GC-related targets were screened from public databases. The overlapping targets between the two were confirmed using a Venn diagram, and a "Resveratrol-Target-GC" network was constructed using Cytoscape software version 3.9.1. The protein-protein interaction (PPI) network was constructed using STRING database and core targets were identified by PPI network analysis. The Database for Annotation, Visualization and Integrated Discovery database was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A "Target-Pathway" network was created by using Cytoscape 3.9.1. The RNA and protein expression levels of core target genes were observed using the Cancer Genome Atlas and the Human Protein Atlas databases. DriverDBv3 and Timer2.0 databases were used for survival and immune infiltration analysis. Subsequently, the findings were further verified by molecular docking technology and in vitro experiments. A total of 378 resveratrol action targets and 2154 GC disease targets were obtained from public databases, and 181 intersection targets between the two were screened by Venn diagram. The top 20 core targets were identified by PPI network analysis of the overlapping targets. GO function analysis mainly involved protein binding, identical protein binding, cytoplasm, nucleus, negative regulation of apoptotic process and response to xenobiotic stimulus. KEGG enrichment analysis suggested that the involved signaling pathways mainly included PI3K-AKT signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, TNF signaling pathway, ErbB signaling pathway, etc. FBJ murine osteosarcoma viral oncogene homolog (FOS) and matrix metallopeptidase 9 (MMP9) were selected by differential expression analysis, and they were closely associated with immune infiltration. Molecular docking results showed that resveratrol docked well with these two targets. Resveratrol treatment arrested the cell cycle at the S phase, induced apoptosis, and weakened viability, migration and invasion in a dose-dependent manner. Furthermore, resveratrol could exhibit anti-GC effect by regulating FOS and MMP9 expression. The anti-GC effects of resveratrol are related to the inhibition of cell proliferation, migration, invasion and induction of cell cycle arrest and apoptosis by targeting FOS and MMP9.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in different tissues from mice infected by Ascaris suum

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

Network pharmacology-based investigation and experimental validation of the therapeutic potential and molecular mechanism of Danshen Chuanxiongqin injection in acute pancreatitis.

Danshen Chuanxiong Injection (DCI) has demonstrated significant clinical efficacy in the treatment of acute pancreatitis (AP); however, the precise molecular mechanisms underlying its therapeutic effects remain incompletely understood. In this study, we employed network pharmacology analysis to comprehensively investigate the active components, potential targets, and signaling pathways involved in DCI-mediated treatment of AP. We utilized the mouse pancreatic acinar cell line 266-6 to establish an cholecystokinin (CCK)-induced AP cell injury model and evaluated cell viability using the Cell counting kit-8 assay. Western blotting and quantitative PCR were employed to determine the expression levels of key target proteins and genes. Network pharmacology analysis identified a total of 144 active components and 430 potential targets within DCI. By integrating data from public databases, we identified 762 AP-related genes. Among these, we identified 93 potential targets that may be involved in the therapeutic effects of DCI for AP. These targets were significantly enriched in biological processes such as oxidative stress, regulation of cytokine production, leukocyte migration, and the TNF signaling pathway. Molecular docking studies revealed a high binding affinity between the active components and the key targets AKT1 and NFKBA, indicative of potential interaction. Additionally, CCK-induced acinar cell injury led to upregulation of AKT1, NFKBA, and P53 proteins, as well as TNF, IL6, and MMP9 genes. Conversely, treatment with DCI dose-dependently attenuated CCK-induced acinar cell injury and restored the expression levels of the aforementioned proteins and genes. Overall, this study provides a comprehensive understanding of the molecular mechanisms underlying the therapeutic effects of DCI in the treatment of AP. Our findings confirm the protective effect of DCI against CCK-induced acinar cell injury and its regulation of key targets.

The involvement of RNA N6-methyladenosine and histone methylation modification in decidualization and endometriosis-associated infertility.

Defective decidualization of endometrial stromal cells (ESCs) in endometriosis (EM) patients leads to inadequate endometrial receptivity and EM-associated infertility. Hypoxia is an inevitable pathological process of EM and participates in deficient decidualization of the eutopic secretory endometrium. Enhancer of zeste homology 2 (EZH2) is a methyltransferase which catalyses H3K27Me3, leading to decreased expression levels of target genes. Although EZH2 expression is low under normal decidualization, it is abundantly increased in the eutopic secretory endometrium of EM and is induced by hypoxia. Chromatin immunoprecipitation-PCR results revealed that decidua marker IGFBP1 is a direct target of EZH2, partially explaining the increased levels of histone methylation modification in defected decidualization of EM. To mechanism controlling this, we examined the effects of hypoxia on EZH2 and decidualization. EZH2 mRNA showed decreased m6 A modification and increased expression levels under hypoxia and decidualization combined treatment. Increased EZH2 expression was due to the increased expression of m6 A demethylase ALKBH5 and decreased expression of the m6 A reader protein YTHDF2. YTHDF2 directly bind to the m6 A modification site of EZH2 to promote EZH2 mRNA degradation in ESCs. Moreover, selective Ezh2 depletion in mouse ESCs increased endometrial receptivity and improved mouse fertility by up-regulating decidua marker IGFBP1 expression. This is the first report showing that YTHDF2 can act as a m6 A reader to promote decidualization by decreasing the stability of EZH2 mRNA and further increasing the expression of IGFBP1 in ESCs. Taken together, our findings highlight the critical role of EZH2/H3K27Me3 in decidualization and reveal a novel epigenetic mechanism by which hypoxia can suppress EM decidualization by decreasing the m6 A modification of EZH2 mRNA.

Resolvin D1 (Rvd1) Attenuates In Vitro LPS-Stimulated Inflammation Through Downregulation of miR-155, miR -146, miR -148 and Krupple Like Factor 5.

Chronic inflammation is associated with many inflammatory diseases. Specialized pro-resolving mediators (SPMs) are well known for their crucial role in promoting the resolution phase of inflammation and restoring tissue homeostasis. Resolvin D1 (RvD1) is an endogenous omega-3-derived lipid mediator with pro-resolving activity. This study aimed to evaluate the effect of Resolvin D1 (RvD1) on some inflammatory miRNAs (mir-155-5p, miR146a-5p and miR148-3p) and Krüppel-like factors 5 (KLF5) in an LPS-stimulated THP-1 preclinical model of inflammation. PMA-differentiated THP-1 cells (macrophages) were pre-incubated with or without various concentrations of RvD1 (10, 50, or 100 nM) for 2 h prior to stimulation by 1 μg/ml LPS. Un-stimulated PMA-differentiated THP-1 cells were as the control group. Then, the expression levels of target genes were evaluated by real-time PCR. Compared with untreated macrophages, stimulation with 1 µg/ml LPS increased mRNA expression levels of TNF-α, KLF5, miR-155-5p, miR-146-5p, and miR-148a-3p. When the cells were exposed to various concentrations (10, 50 and 100 nM) of RvD1 for 2 h prior to LPS stimulation, the TNF-α, KLF5, miR-155-5p, miR-146-5p, and miR-148a-3p mRNA expression levels were significantly downregulated in a dose-dependent manner, compared to the LPS group. The results demonstrate that RvD1 can attenuate inflammatory response in LPS-stimulated macrophages. Our data also showed that RvD1 may exert anti-inflammatory effects by inhibiting miR-155-5p, miR-146a-5p, and miR-148-3p.

MiR-6881-3p contributes to diminished ovarian reserve by regulating granulosa cell apoptosis by targeting SMAD4

BackgroundIn our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood.ObjectiveThis study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR.Materials and methodsInitially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes.ResultsOur findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3’-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased.ConclusionThis study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.

Advanced network pharmacology and molecular docking-based mechanism study to explore the multi-target pharmacological mechanism of Cymbopogon citratus against Alzheimer's disease

BackgroundAlzheimer's disease (AD) is the most common type of dementia, accounting for at least two-thirds of cases of dementia in people age 65 and older. The limited amount of available data and multiple spectra of pathophysiological mechanisms of AD make it a challenging task and a serious economic load on the community health sector. Cymbopogon citratus is a tall perennial fast-growing grass with tuft of lemon-scented leaves from the annulate and sparingly branched rhizomes. A list of studies evidences the therapeutic efficacy of C. citratus against AD, but the precise molecular mechanism is yet to be discovered. Aim of the studyThis research utilizes network pharmacology and molecular docking approaches to elucidate the multi-target effects of C. Citratus on AD, identifying its active compounds, potential therapeutic targets, and associated signaling pathways. Materials and methodsInitially the active compounds of C. citratus, the target genes related to both the C. citratus and the AD were retrieved from literature as well as databases. The PPI network of these target genes was constructed using STRING database and later the hub genes were screened by importing the network into the Cytoscape. The interactions among the target genes and the compounds were also analyzed using Cytoscape. The Gene Ontology (GO) and KEGG pathways of these hub genes were explored to reveal the genes involved in AD-related pathways. These compound-target, genes-pathway networks were explored which uncovered that bioactive compounds of C. citratus may serve as a magic bullet against AD by influencing the target genes involved in the disease pathogenesis. Further, the microarray data was analyzed to explore the expression level of core target genes. Lastly, docking analysis further strengthened the current findings by validating the effective activity of the bioactive compounds against putative target genes. ResultsNetwork pharmacology based analysis highlighted the pathways related to AD, the ten target genes, and the compounds involved in the AD disease mechanism. Through the analysis of microarray expression data, the two genes SRC and IL6 were found to have differential expression. Moreover, the molecular docking analysis predicted the string binding affinity among these genes and active compounds. ConclusionIn summary, our study proposed that five key compounds catechin, apigenin, 8-prenylnaringenin, quercetin, and luteolin contributed to the development of AD by affecting IL6 and SRC genes. The overall integration of network pharmacology with molecular docking unveiled the multi-target pharmacological mechanisms of C. citratus against AD. This study provides convincing evidence that C. citratus might partially alleviate the AD and ultimately lays a foundation for further experimental research on the anti-AD activity of C. citratus.

Genetically proxied therapeutic inhibition of lipid-lowering drug targets and risk of rheumatoid arthritis disease: a Mendelian randomization study.

To evaluate the potential impact of consistent use of similar treatments over a long period; it is essential to investigate the potential correlation between genetic variations that influence the expression or function of pharmacological targets for reducing lipid levels and the risk of developing rheumatoid arthritis. We used variants in the following genes to conduct Mendelian randomization analyses: HMGCR (encoding the target for statins), PCSK9 (encoding the target for PCSK9 inhibitors, such as evolocumab and alirocumab), and NPC1L1 (encoding the target for ezetimibe). Data from lipid genetics consortia (173,082 sample size) were used to weight variations according to their correlations with low-density lipoprotein cholesterol (LDL-C). In two large datasets (total n = 19,562 cases, 501,655 controls). We conducted a meta-analysis of Mendelian randomization estimates, weighted by LDL-C levels, on the regional differences in the risk of rheumatoid arthritis using data from two large databases. We approached SMR and IVW-MR analyses to examine the relationship between target gene expression (including HMGCR, PCSK9, and NPC1L1) and LDL-C levels mediated by these genes with RA. The IVW-MR analysis revealed no significant association between genetically predicted LDL-C concentration and the risk of RA (OR = 0.88, 95% CI = 0.59-1.29; OR = 0.91, 95% CI = 0.67-1.23; OR = 0.81, 95% CI = 0.49-1.36; all p > 0.05). Similarly, our findings from the SMR approach provided no evidence to suggest that gene expression of HMGCR, PCSK9, and NPC1L1 was associated with the risk of RA (OR = 0.91, 95% CI = 0.79-1.05, p = 0.207; OR = 0.96, 95% CI = 0.85-1.09, p = 0.493). Our results do not provide evidence to support the hypothesis that reducing LDL-C levels with statins, alirocumab, or ezetimibe effectively prevents the risk of developing RA. However, our study provides valuable insights into the assessment of lipid-lowering agents in RA, which can enhance our understanding of the condition and assist in clinical practice by aiding in the determination and monitoring of RA status to clinical response.

Investigating the Impact of Vitamin A and Amino Acids on Immune Responses in Celiac Disease Patients.

Amino acids (AAs) and vitamin imbalances are observed in celiac disease (CD). This study evaluated the plasma profile of vitamin A and AAs and the expression level of IL-2, IL-4, IL-10, IL-12 and TGFβ in CD patients. A total of 60 children and adults with CD and 40 healthy controls (HCs) were included. The plasma profile of Vitamin A and AAs and the mRNA expression levels of target genes were assessed. Active adult patients exhibited a decrease in Vitamin A levels (p = 0.04) and an increase in IL-2 (p = 0.008) and IL-12 (p = 0.007) mRNA expression compared to the HCs. The treated adult patients showed elevated Serine (p = 0.003) and Glycine (p = 0.04) levels, as well as increased IL-12 (p < 0.0001) mRNA expression, and a decrease in Tryptophan (p = 0.04) levels relative to the controls. Additionally, the treated adult patients had higher plasma levels of Threonine compared to both the active (p = 0.04) and control (p = 0.02) subjects, and the increased mRNA expression of IL-4 (p = 0.01) in comparison to the active patients. In active children with CD, the IL-2 mRNA level was found to be higher than in the controls (p < 0.0001) and in the treated children (p = 0.005). The treated children with CD exhibited decreased plasma levels of Tryptophan (p = 0.01) and Isoleucine (p = 0.01) relative to the controls, and the increased mRNA expression of TGFβ (p = 0.04) relative to the active patients. Elevated levels of specific AAs (Serine, Glycine, Threonine) in the treated CD patients suggested their potential to improve intestinal damage and inflammation, while decreased levels of Tryptophan and Isoleucine highlighted the need for dietary intervention.

GNAT toxin may have a potential role in Pseudomonas aeruginosa persistence: an invitro and in silico study.

Aims: Persistent cells are primarily responsible for developing antibiotic resistance and the recurrence of Pseudomonas aeruginosa. This study investigated the possible role of GNAT toxin in persistence. Materials & methods: P. aeruginosa was exposed to five MIC concentrations of ciprofloxacin. The expression levels of target genes were assessed. The GNAT/HTH system was bioinformatically studied, and an inhibitory peptide was designed to disrupt this system. Results: Ciprofloxacin can induce bacterial persistence. There was a significant increase in the expression of the GNAT toxin during the persistence state. A structural study of the GNAT/HTH system determined that an inhibitory peptide could be designed to block this system effectively. Conclusion: The GNAT/HTH system shows promise as a novel therapeutic target for combating P. aeruginosa infections.

Controlling gene-expression variability via sequestration-based feedbacks

Expressed Transcription Factors (TFs) not only bind to sites at target promoters but also to decoy sites scattered across the genome. Binding to such “decoys” sequesters TFs critically impacting the response time and stochasticity (noise) in TF and target gene expression level. When the TF is a stable molecule, whose concentration is diluted by cellular growth, our results show that for fixed mean concentration levels, such decoy bindings can both enhance or suppress random fluctuations in TF levels depending on the source of noise (i.e., intrinsic vs. extrinsic noise) and the strength of binding (i.e., weak vs. strong decoys). We implement negative autoregulation where free (unbound) TF molecules inhibit their synthesis. Our analytical results corroborated by numerical simulations reveal that sequestration accentuates the effects of feedback in the sense that noise attenuation by negative feedback is higher with sequestration than in the absence of feedback. We next consider an alternative form of feedback where the TF increases the production of its decoys, and such feedback architectures are frequently seen in endogenous gene regulation involving microRNA-TF circuits and in controlling cellular stress responses. For these circuits where decoy numbers are TF-regulated, we identify limits of noise suppression, and in many cases, these limits occur at intermediate TF-decoy binding affinities.

Cloning and functional analysis of flavanone synthase Ⅱ from Dendrobium huoshanense

Flavonoid C-glycosides are a class of natural products that are widely involved in plant defense responses and have diverse pharmacological activities. They are also important active ingredients of Dendrobium huoshanense. Flavanone synthase Ⅱ has been proven to be a key enzyme in the synthesis pathway of flavonoid C-glycosides in plants, and their catalytic product 2-hydroxyflavanone is the precursor compound for the synthesis of various reported flavonoid C-glycosides. In this study, based on the reported amino acid sequence of flavanone synthase Ⅱ, a flavanone synthase Ⅱ gene(DhuFNSⅡ) was screened and verified from the constructed D. huoshanense genome localization database. Functional validation of the enzyme showed that it could in vitro catalyze naringenin and pinocembrin to produce apigenin and chrysin, respectively. The open reading frame(ORF) of DhuFNSⅡ was 1 644 bp in length, encoding 547 amino acids. Subcellular localization showed that the protein was localized on the endoplasmic reticulum. RT-qPCR results showed that DhuFNSⅡ had the highest expression in stems, followed by leaves and roots. The expression levels of DhuFNSⅡ and other target genes in various tissues of D. huoshanense were significantly up-regulated after four kinds of abiotic stresses commonly encountered in the growth process, but the extent of up-regulation varied among treatment groups, with drought and cold stress having more significant effects on gene expression levels. Through the identification and functional analysis of DhuFNSⅡ, this study is expected to contribute to the elucidation of the molecular mechanism of the formation of quality metabolites of D. huoshanense, flavonoid C-glycosides, and provide a reference for its quality formation and scientific cultivation.

Mechanism analysis of Buyang Huanwu decoction in treating atherosclerosis based on network pharmacology and invitro experiments.

Atherosclerosis (AS) is one of the main risk factors of ischemic cardiovascular and cerebrovascular diseases. Buyang Huanwu decoction (BYHWT) is a classic Chinese medicine prescription that is used for treating AS. However, the underlying pharmacological mechanism remains unclear. This study aims to clarify the molecular mechanism of BYHWT in treatment of AS through network pharmacology and invitro experiments. Molecular structure information and targets of core components of BYHWT were obtained from PubChem and UniProtKB databases. Genes involved in AS were obtained from DisGeNet, GeneCards and OMIM databases. The core targets of BYHWT in AS treatment were identified by protein-protein interaction (PPI) network analysis with STRING platform, and analyzed by gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis. Molecular docking was used to verify the binding affinity between the core targets and the bioactive ingredients. HUVEC viability, inflammatory response and mRNA expression levels of core target genes were evaluated by cell counting kit 8 assay, enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. A total of 60 candidate compounds and 325 predicted target genes were screened. PPI network analysis suggested that TP53, SRC, STAT3, and AKT1 may be the core targets. BYHWT in AS treatment was associated with 46 signaling pathways. GA120, baicalein, and 3,9-di-o-methylnissolin had good binding affinity with core target proteins. Baicalein treatment could significantly promoted the viability and repress the inflammatory response of HUVEC cells stimulated by ox-LDL. In addition, Baicalein can regulate the expression of core targets including AKT1, MAPK1, PIK3CA, JUN, TP53, SRC, EGFR, and ESR1. In conclusion, BYHWT and its main bioactive component baicalein, inhibit inflammatory response and modulate multiple downstream genes of endothelial cells, and show good potential to block the progression of AS and cardiovascular/cerebrovascular diseases.

Association of MIR4435-2HG Expression with TP53 Mutation, Estrogen Receptor Activity, and Poor Prognosis in Breast Cancer

Background: The roles of many long noncoding RNAs (lncRNAs) in breast cancer (BC) are unclear, and the medications that affect them have attracted less attention. Objectives: This study aimed to identify lncRNAs that have the highest association with poor prognosis in BC. Moreover, the relationship between the expression of identified lncRNA and conventional chemotherapy agents was investigated. Methods: The cancer genome atlas data were used to identify lncRNA with the most significant impact on BC survival. The relation of identified lncRNA levels with different subtypes of BC and mutation was assessed. PharmacoGX data and the GEO database were used to evaluate the association of identified lncRNA levels with drug sensitivity and find appropriate medications to reduce its expression, respectively. To assess the effect of the identified drug on the level of lncRNA expression, MCF7, and MDA-MB-468 cell lines were used, and the expression level of target genes was assessed by quantitative reverse transcription polymerase chain reaction. Results: The expression level of lncRNA MIR4435-2HG was most associated with the poor survival of BC patients, and its expression level can be related to TP53 mutation and estrogen and progesterone receptors. Increased expression of MIR4435-2HG was observed in cancer samples, and MIR4435-2HG level was correlated with metastasis pathway genes. Drug sensitivity to epirubicin, irinotecan, and lestaurtinib correlated with MIR4435-2HG levels. Furthermore, GEO data showed that the expression level of MIR4435-2HG could decrease under estrogen receptor (ER) activity. Finally, in vitro, results showed that MIR4435-2HG expression declined in response to estradiol as an ER-activating ligand in the MCF7 cell line. Conclusions: The expression of MIR4435-2HG can be a powerful biomarker for poor prognosis in BC. The data also suggested that this lncRNA may play an oncogenic role in metastatic pathways, and ER activity could regulate its expression.

MiR-221-3p/222-3p Cluster Expression in Human Adipose Tissue Is Related to Obesity and Type 2 Diabetes.

The progression of obesity and type 2 diabetes (T2D) is intricately linked with adipose tissue (AT) angiogenesis. Despite an established network of microRNAs (miRNAs) regulating AT function, the specific role of angiogenic miRNAs remains less understood. The miR-221/222 cluster has recently emerged as being associated with antiangiogenic activity. However, no studies have explored its role in human AT amidst the concurrent development of obesity and T2D. Therefore, this study aims to investigate the association between the miR-221-3p/222-3p cluster in human AT and its regulatory network with obesity and T2D. MiR-221-3p/222-3p and their target gene (TG) expression levels were quantified through qPCR in visceral (VAT) and subcutaneous (SAT) AT from patients (n = 33) categorized based on BMI as normoweight (NW) and obese (OB) and by glycemic status as normoglycemic (NG) and type 2 diabetic (T2D) subjects. In silico analyses of miR-221-3p/222-3p and their TGs were conducted to identify pertinent signaling pathways. The results of a multivariate analysis, considering the simultaneous expression of miR-221-3p and miR-222-3p as dependent variables, revealed statistically significant distinctions when accounting for variables such as tissue depot, obesity, sex, and T2D as independent factors. Furthermore, both miRNAs and their TGs exhibited differential expression patterns based on obesity severity, glycemic status, sex, and type of AT depot. Our in silico analysis indicated that miR-221-3p/222-3p cluster TGs predominantly participate in angiogenesis, WNT signaling, and apoptosis pathways. In conclusion, these findings underscore a promising avenue for future research, emphasizing the miR-221-3p/222-3p cluster and its associated regulatory networks as potential targets for addressing obesity and related metabolic disorders.

GDF-10 Induces an Inhibitory Effect on Epithelial-Mesenchymal Transition of Laryngeal Cancer via LPR4.

Growth differentiation factor-10 (GDF-10), a member of the TGF-β superfamily, plays a crucial role in cell proliferation and differentiation. In some tumors, GDF-10 can act as a tumor suppressor to inhibit tumor progression, but its role in posterior squamous cell carcinoma has not been reported yet. The aim of this study was to investigate the effect of GDF-10 on the epithelial-mesenchymal transition of laryngeal squamous cell carcinoma, and to provide new ideas for future targets in the treatment of laryngeal squamous carcinoma. The effect of GDF-10 on tumor growth was detected; bioinformatics analysis was performed to predict the downstream targets of GDF-10, and RT-PCR and western blot were performed to detect the expression levels of target genes and proteins, respectively. Our findings support that GDF-10 can inhibit the proliferation, migration, and invasion, and promote apoptosis of laryngeal carcinoma AMC-HN-8 cells. GDF-10 inhibits the EMT of laryngeal carcinoma through LRP4 and thus inhibits the progression of laryngeal carcinoma.

Abstract A056: Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer

Abstract The Androgen Receptor (AR) cistrome is critical in the development of prostate cell identity while its misregulation promotes prostate cancer development. The pioneer transcription factor Forkhead box A1 (FOXA1) has been shown to be essential for the recruitment of AR to androgen response elements (AREs) allowing for reprogramming of the AR cistrome resulting in prostate cell transformation. FHD-286 is a BRM/BRG1 dual ATPase inhibitor currently in clinical trials for AML. Here, we show that treatment of prostate cancer cell lines with FHD-286 results in ablation of FOXA1 mediated AREs at tumor-associated AR binding sites (T-ARBS). Dual ATPase treatment subsequently reduces the expression level of various oncogenic AR target genes, leading to a decrease in tumor cell viability. Both patient derived organoid and In vivo studies provide further validation by showing a decrease in tumor growth. Strikingly, inhibition of BAF complex activity bypasses AR resistance mechanisms commonly seen after castration and Enzalutamide treatment as cell lines containing AR-V7 splice variants and neuroendocrine organoids display sensitivity. Taken together, our data illustrates a novel mechanism of treating AR mediated prostate cancer though the inhibition tumor associated AREs by treatment with FHD-286. Citation Format: Gabriel J Sandoval, Richard C Centore, Sal Topal, Dave Lahr, GiNell Elliott, Heena Gandevia, Ammar Adam, Jessica Piel, Holly M Nguyen, Eva Corey, Yu Chen, Steven Bellon, Murphy Hentemann. Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A056.

Alternatively Spliced ATP6V1H induces Inflammation in Polycythemia Vera and Essential Thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are clonal myeloproliferative neoplasm originating from a hematopoietic stem cell. PV is characterized by increased hemoglobin; ET is characterized by high platelets due to gain-of-function mutations of the tyrosine kinase JAK2 gene, with JAK2V617F being the most common phenotype defining mutation. Thrombosis remains a major cause of PV and ET morbidity and mortality. Clonal myelopoiesis in PV is further enhanced by inflammation, contributing to PV pathogenesis. The molecular mechanisms underlying the augmented inflammation in PV remain incompletely characterized but neutrophils play a major role in inflammatory and prothrombotic complications in PV and ET. Our whole transcriptome analysis using 46 PV neutrophils and 10 healthy controls showed that 1833 genes in PV granulocytes (adjusted p-value &amp;lt;0.05 and log2 fold changes &amp;gt;0.5) were dysregulated. Gene set enrichment analysis showed that genes involved in inflammatory response were upregulated in PV (FDR: 0.047, enrichment score: 0.578). We then interrogated in our transcriptome set differential exon usage and identified a differentially spliced exon of ATP6V1H (AS-ATP6V1H) in PV. ATP6V1H encodes ATPase H+ transporting V1 (v-ATPase) subunit H. Inhibition of v-ATPase augments the production, release, and expression of inflammatory genes. We found higher expression levels of AS-ATP6V1H (ENST00000522849) in PV neutrophil transcriptome (Figure B). The exon in the black box in Figure A is only present in AS-ATP6V1H (ENST00000522849) and its expression was higher in PV. This transcript undergoes nonsense-mediated decay, resulting in decreased ATP6V1H expression in PV (Figure C). This finding was then verified using 46 patients with PV, 24 with ET and 29 healthy controls. AS-ATP6V1H was upregulated both in PV and ET neutrophils (Figures D). However, these expression differences were not detected in platelets (Figure E). AS-ATP6V1H transcript levels demonstrated a positive correlation with genes that are upregulated in inflammation - CXCL8, CD55, IRAK1, and IL1RAP, while showing an inverse correlation with genes downregulated in inflammation, including IGHG1, CD40LG, and TNFRSF4.These transcript levels showed a positive correlation with white blood cell (r=0.4677, p&amp;lt;0001) and neutrophil counts (r=0.5268, p&amp;lt;0001), indicating an association with heightened inflammation in PV and ET. PV and ET patients with a history of thrombosis had higher AS-ATP6V1H transcript levels compared to those without thromboses (p=0.0427). AS-ATP6V1H transcript levels positively correlated with the levels of thrombotic genes tissue factor ( F3) and p-selectin ( SELP) and inversely correlated with an anti-thrombotic gene, KLF2 (Song, Blood Advances, 2023). These results suggest that elevated AS-ATP6V1H may also be associated with an increased risk of thrombosis. The expression of inflammatory and thrombotic genes is influenced by hypoxia inducible factors (HIFs) (Gangaraju, Blood Advances, 2020). Reduced ATP6V1H increases HIF-1a protein levels and its transcriptional activities (Yambire, elife, 2019). We also measured the expression levels of HIF target genes in neutrophils ( EDN1, LDHA, SLC29A1, CKB, VEGFA, and SLC2A1), and these levels positively correlated with AS-ATP6V1H transcript levels. Since inflammation is also associated with increasing JAK2V617F variant allele frequency (VAF), we also tested the correlation with JAK2V617F VAF. We observed a positive correlation of JAK2V617F VAF with AS-ATP6V1H transcript levels (r=0.74, p&amp;lt;0001), suggesting that hyperactive JAK2 activity might induce an mRNA splicing event in ATP6V1H. In conclusion, our study showed a relationship between AS-ATP6V1H and inflammation in PV and ET. Through whole transcriptome analysis, we identified AS-ATP6V1H as a candidate gene, and its upregulation in PV and ET suggests its potential involvement in increased inflammation and clonal myelopoiesis. We show that AS-ATP6V1H transcript levels correlated with the expression levels of genes involved in inflammation and thrombosis and that AS-ATP6V1H may be regulated by HIFs and JAK2 activity. Further investigations should demonstrate the molecular mechanisms underlying AS-ATP6V1H's role in inflammation and thrombosis, which could lead to novel therapeutic approaches for managing PV and ET.