Cytokine-mediated Signaling Pathway Research Articles

Fatty acid synthase (Fasn) inhibits the expression levels of immune response genes via alteration of alternative splicing in islet cells

BackgroundIncreasing evidence has shown that fatty acid synthase (Fasn) is associated with diabetes mellitus (DM) and insulin resistance, however, it remains unclear how Fasn upregulation leads to dysregulation of energy homeostasis in islet cells. Consequently, uncovering the function of Fasn in islet cells. Consequently, uncovering the function of FASN in islet cells is immensely important for finding a treatment target. AimIn this study, we elucidated the biological function of Fasn on the target genes in a rat insulinoma INS-1 cell line. MethodsWe created a Fasn overexpressing rat insulinoma cell line (Fasn-OE), and performed bulk RNA-sequencing (RNA-seq) experiments on Fasn-OE and INS-1 (control) cells. We first identified differentially expressed genes (DEGs) using Bioconductor package edgeR, and then discovered enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the KEGG Orthology Based Annotation System (KOBAS) 2.0 web server. Furthermore, we identified alternative splicing events (ASEs) and regulated alternative splicing events (RASEs) by applying the ABLas pipeline. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used for validation of selected differentially expressed genes (DEGs) and Fasn-regulated alternative splicing genes (RASGs). ResultsIn this study we found that Fasn overexpression led to significant changes of gene expression profiles, including downregulations of mRNA levels of immune related genes, including Bst2, Ddit3, Isg15, Mx2, Oas1a, Oasl, and RT1-S3 in INS-1 cell line. Furthermore, Fasn positively regulated the expression of transcription factors such as Fat1 and Ncl diabetes-related genes. Importantly, Fasn overexpression to result in alternative splicing events including in a metabolism-associated ATP binding protein mRNA Abcc5. In Gene Ontology analysis, the downregulated genes in Fasn-OE cells were mainly enriched in inflammatory response and innate immune response. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the downregulated genes were mainly enriched in TNF signaling pathway and cytokine-mediated signaling pathways. ConclusionsOur findings showed that upregulation of Fasn may play a critical role in islet cell immunmetabolism via modifications of immune/inflammatory related genes on transcription and alternative splicing level, which provide novel insights into characterizing the function of Fasn in islet cell immunity and for the development of chemo/immune therapies.

Comprehensive Network Analysis Identified SIRT7, NTRK2, and CHI3L1 as New Potential Markers for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is considered the basis of serious clinical symptoms, especially for low back pain (LBP). Therefore, it is essential to explore the regulatory role and diagnostic performance of dysregulated genes and potential drugs in IDD. Through WGCNA co-expression analysis, 36 co-expression modules were obtained. Among them, MidnightBlue and Red modules were the most related to IDD. Functional enrichment analysis showed that the Red module was mainly related to neutrophil activation and regulation of cytokine-mediated signaling pathway and apoptosis, whereas the MidnightBlue module was mainly related to extracellular matrix organization, bone development, extracellular matrix, extracellular matrix component, and other extracellular matrices. Furthermore, 356 genes highly related to the module were screened to construct a protein interaction network. Network degree distribution analysis showed that the known IDD-related genes had a higher degree of distribution. Enrichment analysis demonstrated that these genes were enriched in MAPK_SIGNALING_PATHWAY (FDR = 0.012), CHEMOKINE_SIGNALING_PATHWAY, and some other pathways. By constructing a disease-gene interaction network, three disease-specific genes were finally identified. Through combining with the drug-target gene interaction network, two potential therapeutic drugs, entrectinib and larotrectinib, were determined. Finally, based on these genes, the diagnostic model in the training dataset, test dataset, and verification dataset all showed a high diagnostic performance. The findings of this study contributed to the diagnosis of IDD and personalized treatment of IDD.

An Integrative Network Approach to Identify Common Genes for the Therapeutics in Tuberculosis and Its Overlapping Non-Communicable Diseases.

Tuberculosis (TB) is the leading cause of death from a single infectious agent. The estimated total global TB deaths in 2019 were 1.4 million. The decline in TB incidence rate is very slow, while the burden of noncommunicable diseases (NCDs) is exponentially increasing in low- and middle-income countries, where the prevention and treatment of TB disease remains a great burden, and there is enough empirical evidence (scientific evidence) to justify a greater research emphasis on the syndemic interaction between TB and NCDs. The current study was proposed to build a disease-gene network based on overlapping TB with NCDs (overlapping means genes involved in TB and other/s NCDs), such as Parkinson’s disease, cardiovascular disease, diabetes mellitus, rheumatoid arthritis, and lung cancer. We compared the TB-associated genes with genes of its overlapping NCDs to determine the gene-disease relationship. Next, we constructed the gene interaction network of disease-genes by integrating curated and experimentally validated interactions in humans and find the 13 highly clustered modules in the network, which contains a total of 86 hub genes that are commonly associated with TB and its overlapping NCDs, which are largely involved in the Inflammatory response, cellular response to cytokine stimulus, response to cytokine, cytokine-mediated signaling pathway, defense response, response to stress and immune system process. Moreover, the identified hub genes and their respective drugs were exploited to build a bipartite network that assists in deciphering the drug-target interaction, highlighting the influential roles of these drugs on apparently unrelated targets and pathways. Targeting these hub proteins by using drugs combination or drug repurposing approaches will improve the clinical conditions in comorbidity, enhance the potency of a few drugs, and give a synergistic effect with better outcomes. Thus, understanding the Mycobacterium tuberculosis (Mtb) infection and associated NCDs is a high priority to contain its short and long-term effects on human health. Our network-based analysis opens a new horizon for more personalized treatment, drug-repurposing opportunities, investigates new targets, multidrug treatment, and can uncover several side effects of unrelated drugs for TB and its overlapping NCDs.

EPITHELIAL DUOX2 ACTIVATION INCREASES GUT PERMEABILITY AND BACTERIAL TRANSLOCATION THAT IS RESCUED WITH BUTYRATE SUPPLEMENTATION

Abstract BACKGROUND Inflammatory bowel diseases (IBD) are chronic pathologies characterized by dysbiosis, defects in epithelial barrier function, and increased redox stress. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, which produce metabolites important in maintaining gut homeostasis. The epithelial NADPH oxidase dual oxidase 2 (DUOX2) catalyzes the production of hydrogen peroxide (H2O2) and is the only gene consistently altered in IBD patients in association with dysbiosis and before the onset of disease. However, the functional consequence of DUOX2 activation in IBD is not understood. Using models of dysbiosis leading to DUOX2 activation, we aimed to dissect the contribution of DUOX2 activity to the defects in epithelial barrier function observed in IBD. METHODS villin-TLR4 (vTLR4) mice, which express a constitutively active form of TLR4 in colonic epithelial cells (CECs) and have increased activation of DUOX2, vTLR4-DUOX2-KO mice, and their wild-type (WT) littermates were euthanized. Livers were collected for permeability assessment by bacterial translocation. Colonic tissue was collected for RNA-sequencing and isolation of CECs for gene expression determinations by qPCR. Separately, vTLR4 mice and littermates were treated with butyrate in drinking water. Tissue was harvested for the same determinations and for measurement of epithelial H2O2 production via Amplex Red. Colonoids from WT mice were treated with butyrate and stimulated with IFNγ or heat-killed adherent invasive Escherichia coli. H2O2 production and gene expression of Duox2 were determined. RESULTS RNA-sequencing revealed that abrogation of DUOX2 activity downregulated cytokine-mediated signaling and cytokine production pathways, including tumor necrosis factors and key chemotactic proteins such as CXCL1 and CXCL2. Similarly, expression of Tnfa, Cxcl1, and Cxcl2 was significantly downregulated to WT levels in vTLR4-DUOX2-KO mice. vTLR4 mice had increased bacterial translocation, which was rescued by deletion of DUOX2. Butyrate treatment inhibited epithelial H2O2 response to stimuli in vitro, which was accompanied by a reduction in Duox2 transcripts. In vivo, butyrate treatment of vTLR4 mice significantly reduced epithelial production of H2O2, bacterial translocation to the liver, and led to a downregulation of Tnfa, Cxcl1, and Cxcl2 transcripts. CONCLUSIONS Chronic DUOX2 activity leads to defects in epithelial barrier function resulting in increased bacterial translocation. This leads to an increase in proinflammatory cytokines and chemokines. The microbial metabolite butyrate blocked DUOX2 activity, rescuing intestinal permeability and reducing bacterial translocation and inflammatory cytokine gene expression. We posit that therapies aimed at controlling DUOX2 activity through microbiome or metabolome-targeted approaches may be beneficial in IBD.

Molecular docking and pharmacology study to explore bio-active compounds and underlying mechanisms of Caesalpinia bonducella on polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) was effectively treated with Caesalpinia bonducella (CB) although its active components and molecular mechanisms remain undetermined. Through network pharmacology and molecular docking technology, the ultimate goal of this study was to investigate the active ingredients and functions of CB against PCOS. A literature review and PhytochemDB database were used to identify the compounds in CB which were then subjected to an ADMET (Absorption, Distribution, Metabolism, Elimination and Toxicity) assessment. Then, the identified common genes were used for network construction through Enrichr and STRING database and docking were performed in AutoDock software. During ADMET screening, only 19 compounds showed excellent potential biological activity out of 51 active ingredients of CB. Furthermore, we have identified 137 and 1976 genes for target compounds and PCOS, respectively by using several public databases. The findings of gene-compounds network analysis revealed that the active ingredients and target genes of CB against PCOS comprised of 5 phytochemicals and 42 overlapping genes, among these flavone and IL-6 being the top active ingredient and hub proteins, respectively. From the pathway enrichment analysis, cytokine-mediated, IL-17, and TLR signaling pathways were the most significant. PPI and molecular docking analysis indicated that CAT -9.3 kcal/mol, TNF (−8.4 kcal/mol) and IL-6 (−8.3 kcal/mol) proteins were the potential biomarkers of PCOS pathogenesis compared to control drug (clomifene citrate). Applying network pharmacology and molecular docking, the active components and molecular mechanisms of CB against PCOS were initially studied. Our study provides scientific evidence that may support the therapeutic impacts of CB and need to be further validation of active compounds in treatment of PCOS.

Repression of the expression of proinflammatory genes by mitochondrial transcription factor A is linked to its alternative splicing regulation in human lung epithelial cells

BackgroundMitochondrial transcription factor A (TFAM) is associated with a number of neurodegenerative diseases and also with asthma. TFAM deficiency-induced mitochondrial DNA stress primes the antiviral innate immune response in mouse embryonic fibroblasts. However, the role of TFAM in asthma related inflammation remains obscure. The purpose of this study was to investigate the regulatory mechanism of TFAM in asthma.ResultsIn this study, we overexpressed TFAM in human lung epithelial cells (A549), then obtained the TFAM-regulated transcriptome by Illumina sequencing technology. Transcriptome analysis revealed that TFAM overexpression down-regulated and up-regulated the expression of 642 and 169 differentially expressed genes (DEGs), respectively. The TFAM-repressed genes were strongly enriched in cytokine-mediated signaling pathway, type I interferon- and INF-γ-mediated signaling pathways, and viral response pathways. We also revealed that 2563 alternative splicing events in 1796 alternative splicing genes (ASGs) were de-regulated upon TFAM overexpression. These TFAM-responding ASGs were enriched in DNA repair, nerve growth factor receptor signaling pathway, and also transcription regulation. Further analysis revealed that the promoters of TFAM-repressed DEGs were enriched by DNA binding motifs of transcription factors whose alternative splicing was regulated by TFAM.ConclusionsThese findings suggest that TFAM regulates not only immune response gene expression in human lung epithelial cells, but also pre-mRNA alternative splicing which may mediate transcriptional regulation; this TFAM-centered gene regulation network could be targeted in developing therapies against various diseases.

Transcriptomic profiling reveals gene expression in human peripheral blood after exposure to low-dose ionizing radiation.

Although the health effects of exposure to low-dose ionizing radiation have been the focus of many studies, the affected biological functions and underlying regulatory mechanisms are not well-understood. In particular, the influence of radiation exposure at doses of less than 200 mGy on the regulation of genes and pathways remains unclear. To investigate the molecular alterations induced by varying doses of low-dose radiation (LDR), transcriptomic analysis was conducted based on ribonucleic acid (RNA) sequencing following exposure to 50 and 150 mGy doses. Human peripheral blood was collected, and the samples were divided into three groups, including two treatments and one control (no radiation). A total of 876 (318 upregulated and 558 downregulated) and 486 (202 upregulated and 284 downregulated) differentially expressed genes (DEGs) were identified after exposure to 50 mGy and 150 mGy, respectively. Most upregulated genes in both the 50 mGy and 150 mGy groups were associated with ‘antigen processing and presentation,’ which appeared to be the major targets affected by LDR exposure. Several interacting genes, including HLA-DQA1, HLA-DQA2, HLA-DQB2, HLA-DRB1, and HLA-DRB5 were mapped to ‘antigen processing and presentation,’ ‘immune system-related diseases’ and the ‘cytokine-mediated signaling pathway,’ suggesting that these genes might drive the downstream transmission of these signal transduction pathways. Our results suggest that exposure to LDR may elicit changes in key genes and associated pathways, probably helping further explore the biological processes and molecular mechanism responsible for low-dose occupational or environmental exposures in humans.

Identification of Hub Genes in Tuberculosis via Bioinformatics Analysis.

Background Tuberculosis (TB) is a serious chronic bacterial infection caused by Mycobacterium tuberculosis (MTB). It is one of the deadliest diseases in the world and a heavy burden for people all over the world. However, the hub genes involved in the host response remain largely unclear. Methods The data set GSE11199 was studied to clarify the potential gene network and signal transduction pathway in TB. The subjects were divided into latent tuberculosis and pulmonary tuberculosis, and the distribution of differentially expressed genes (DEGs) was analyzed between them using GEO2R. We verified the enriched process and pathway of DEGs by making use of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). The construction of protein-protein interaction (PPI) network of DEGs was achieved through making use of the Search Tool for the Retrieval of Interacting Genes (STRING), aiming at identifying hub genes. Then, the hub gene expression level in latent and pulmonary tuberculosis was verified by a boxplot. Finally, through making use of Gene Set Enrichment Analysis (GSEA), we further analyzed the pathways related to DEGs in the data set GSE11199 to show the changing pattern between latent and pulmonary tuberculosis. Results We identified 98 DEGs in total in the data set GSE11199, 91 genes upregulated and 7 genes downregulated included. The enrichment of GO and KEGG pathways demonstrated that upregulated DEGs were mainly abundant in cytokine-mediated signaling pathway, response to interferon-gamma, endoplasmic reticulum lumen, beta-galactosidase activity, measles, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, etc. Based on the PPI network, we obtained 4 hub genes with a higher degree, namely, CTLA4, GZMB, GZMA, and PRF1. The box plot showed that these 4 hub gene expression levels in the pulmonary tuberculosis group were higher than those in the latent group. Finally, through Gene Set Enrichment Analysis (GSEA), it was concluded that DEGs were largely associated with proteasome and primary immunodeficiency. Conclusions This study reveals the coordination of pathogenic genes during TB infection and offers the diagnosis of TB a promising genome. These hub genes also provide new directions for the development of latent molecular targets for TB treatment.

Expression Profile of New Gene Markers and Signaling Pathways Involved in Immunological Processes in Human Cumulus-Oophorus Cells.

The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus–oocyte complex (COC) show evolving gene expression profiles throughout the oocytes’ lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs’ gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the “defense response to other organism”, “defense response”, “defense response to virus”, “cytokine secretion”, “cytokine production” and “cytokine-mediated signaling pathway” GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells.

Weighted Gene Co-Expression Network Analysis Combined with Machine Learning Validation to Identify Key Modules and Hub Genes Associated with SARS-CoV-2 Infection.

The coronavirus disease-2019 (COVID-19) pandemic has caused an enormous loss of lives. Various clinical trials of vaccines and drugs are being conducted worldwide; nevertheless, as of today, no effective drug exists for COVID-19. The identification of key genes and pathways in this disease may lead to finding potential drug targets and biomarkers. Here, we applied weighted gene co-expression network analysis and LIME as an explainable artificial intelligence algorithm to comprehensively characterize transcriptional changes in bronchial epithelium cells (primary human lung epithelium (NHBE) and transformed lung alveolar (A549) cells) during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study detected a network that significantly correlated to the pathogenicity of COVID-19 infection based on identified hub genes in each cell line separately. The novel hub gene signature that was detected in our study, including PGLYRP4 and HEPHL1, may shed light on the pathogenesis of COVID-19, holding promise for future prognostic and therapeutic approaches. The enrichment analysis of hub genes showed that the most relevant biological process and KEGG pathways were the type I interferon signaling pathway, IL-17 signaling pathway, cytokine-mediated signaling pathway, and defense response to virus categories, all of which play significant roles in restricting viral infection. Moreover, according to the drug–target network, we identified 17 novel FDA-approved candidate drugs, which could potentially be used to treat COVID-19 patients through the regulation of four hub genes of the co-expression network. In conclusion, the aforementioned hub genes might play potential roles in translational medicine and might become promising therapeutic targets. Further in vitro and in vivo experimental studies are needed to evaluate the role of these hub genes in COVID-19.

Use of Network Pharmacology to Investigate the Mechanism of the Compound Xuanju Capsule in the Treatment of Rheumatoid Arthritis.

Objective To clarify the therapeutic mechanisms of compound Xuanju capsule-treated rheumatoid arthritis (RA) based on network pharmacology tactics. Method The TCMSP, TCMID and STITCH databases were used to screen the active ingredients and targets in the compound Xuanju capsule; the OMIM, TTD, PharmGKB and GeneCards databases were applied to screen the RA-related disease targets. Then, the obtained targets were imported into Cytoscape 3.7.1 software to construct the active ingredient-target network and the RA-related disease-target network. The active ingredient-target PPI network, the RA-related disease-target PPI network and the common target PPI network were built by using the STRING platform and Cytoscape 3.7.1 software. The GO and KEGG analyses of the common targets were analyzed by using the Metascape and Bioinformatics online tools. Results A total of 51 active ingredients and 513 corresponding ingredient targets were harvested from the compound Xuanju capsule; 641 RA-related disease targets were obtained. After two PPI networks were constructed and merged, 116 RA-related targets of compound Xuanju capsules were identified and analyzed. 116 RA-related targets of compound Xuanju capsules are mainly involved in the biological processes and molecular functions, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide and the blood vascular development, the cytokine activity, the cytokine receptor binding and the receptor regulator activity. Furthermore, 116 RA-related targets of compound Xuanju capsules are concentrated in signaling pathways such as the IL-17, TNF, Th17 cell differentiation, Toll receptor and RA signaling pathway. Conclusion The compound Xuanju capsule had the action characteristics of multiple components, multiple targets, and multiple pathways in the treatment of RA, which might primarily reduce the release of proinflammatory factors (such as IL-6 and TNF-α) and increase the production of anti-inflammatory factors (such as IL-10) by regulating inflammation-related signaling pathways (such as IL-17), thereby alleviating the inflammatory damage and improving the bone tissue repair.

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

BackgroundOur understanding of adaptive immune responses in patients with coronavirus disease 2019 (COVID-19) is rapidly evolving, but information on the innate immune responses by natural killer (NK) cells is still insufficient.ObjectiveWe aimed to examine the phenotypic and functional status of NK cells and their changes during the course of mild and severe COVID-19.MethodsWe performed RNA sequencing and flow cytometric analysis of NK cells from patients with mild and severe COVID-19 at multiple time points in the course of the disease using cryopreserved PBMCs.ResultsIn RNA-sequencing analysis, the NK cells exhibited distinctive features compared with healthy donors, with significant enrichment of proinflammatory cytokine-mediated signaling pathways. Intriguingly, we found that the unconventional CD56dimCD16neg NK-cell population expanded in cryopreserved PBMCs from patients with COVID-19 regardless of disease severity, accompanied by decreased NK-cell cytotoxicity. The NK-cell population was rapidly normalized alongside the disappearance of unconventional CD56dimCD16neg NK cells and the recovery of NK-cell cytotoxicity in patients with mild COVID-19, but this occurred slowly in patients with severe COVID-19.ConclusionsThe current longitudinal study provides a deep understanding of the NK-cell biology in COVID-19.

Integrated Analysis of lncRNAs, mRNAs, and TFs to Identify Regulatory Networks Underlying MAP Infection in Cattle

Johne’s disease is a chronic infection of ruminants that burdens dairy herds with a significant economic loss. The pathogenesis of the disease has not been revealed clearly due to its complex nature. In order to achieve deeper biological insights into molecular mechanisms involved in MAP infection resulting in Johne’s disease, a system biology approach was used. As far as is known, this is the first study that considers lncRNAs, TFs, and mRNAs, simultaneously, to construct an integrated gene regulatory network involved in MAP infection. Weighted gene coexpression network analysis (WGCNA) and functional enrichment analysis were conducted to explore coexpression modules from which nonpreserved modules had altered connectivity patterns. After identification of hub and hub-hub genes as well as TFs and lncRNAs in the nonpreserved modules, integrated networks of lncRNA-mRNA-TF were constructed, and cis and trans targets of lncRNAs were identified. Both cis and trans targets of lncRNAs were found in eight nonpreserved modules. Twenty-one of 47 nonpreserved modules showed significant biological processes related to the immune system and MAP infection. Some of the MAP infection’s related pathways in the most important nonpreserved modules comprise “positive regulation of cytokine-mediated signaling pathway,” “negative regulation of leukocyte migration,” “T-cell differentiation,” “neutrophil activation,” and “defense response.” Furthermore, several genes were identified in these modules, including SLC11A1, MAPK8IP1, HMGCR, IFNGR1, CMPK2, CORO1A, IRF1, LDLR, BOLA-DMB, and BOLA-DMA, which are potentially associated with MAP pathogenesis. This study not only enhanced our knowledge of molecular mechanisms behind MAP infection but also highlighted several promising hub and hub-hub genes involved in macrophage-pathogen interaction.

Bioinformatics Analysis of Expression Profiles and Prognostic Values of the Signal Transducer and Activator of Transcription Family Genes in Glioma.

Signal transducer and activator of transcription (STAT) family genes—of which there are seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6—have been associated with the progression of multiple cancers. However, their prognostic values in glioma remain unclear. In this study, we systematically investigated the expression, the prognostic value, and the potential mechanism of the STAT family genes in glioma. The expression of STAT1/2/3/5A/6 members were significantly higher and positively correlated with IDH mutations, while the expression of STAT5B was lower and negatively correlated with IDH mutations in glioma. Survival analysis indicated that the upregulation of STAT1/2/3/5A/6 and downregulation of STAT5B expression was associated with poorer overall survival in glioma. Joint effects analysis of STAT1/2/3/5A/5B/6 expression suggested that the prognostic value of the group was more significant than that of each individual gene. Thus, we constructed a risk score model to predict the prognosis of glioma. The receiver operating characteristic curve and calibration curves showed good performance as prognostic indicators in both TCGA (The Cancer Genome Atlas) and the CGGA (Chinese Glioma Genome Atlas) databases. Furthermore, we analyzed the correlation between STAT expression with immune infiltration in glioma. The Protein–protein interaction network and enrichment analysis showed that STAT members and co-expressed genes mainly participated in signal transduction activity, Hepatitis B, the Jak-STAT signaling pathway, transcription factor activity, sequence-specific DNA binding, and the cytokine-mediated signaling pathway in glioma. In summary, our study analyzed the expression, prognostic values, and biological roles of the STAT gene family members in glioma, based on which we developed a new risk score model to predict the prognosis of glioma more precisely.

Abstract 1679: Innate immunity and cytokine signaling pathways drive resistance to HSV-1 oncolytic immunovirotherapy in glioblastoma

Abstract Glioblastoma multiforme (GBM) is a particularly aggressive primary brain tumor that is relatively common in adults. The disease comes with a dismal prognosis, often with expected survival just over a year when treated with surgery, chemotherapy, and radiation. A promising addition to our GBM treatment arsenal are oncolytic viruses (OVs), a class of immunotherapy now in clinical trials. The most effective OVs home in on tumor cells, killing them and releasing tumor-associated antigens that elicit antitumor immunity. This effectiveness, however, varies depending on the cell state—a property that may be influenced by the composition of the extracellular matrix (ECM) of GBM. Cellular communication network factor 1 (CCN1) is found in the ECM of the majority of GBMs and is predictive of resistance to OVs, particularly those derived from herpes simplex virus type 1 (HSV-1). In this study, our aim is to understand how extracellular CCN1 alters the GBM intracellular state in a manner that confers resistance to OVs. We began by using our NetDecoder platform to build CCN1-specific prioritized protein-protein interaction (PPI) networks from published LN229 human GBM transcriptomic datasets. From this network, we identified 12 PPI edges that dominate the network flow in CCN1-high LN229 cells when compared to CCN1-low controls. The 21 unique nodes that comprise these binary PPI edges were extracted and subjected to downstream biological process overrepresentation analyses using Gene Ontology. We found that the cytokine-mediated signaling pathway (FDR = 4.2 x 10-11; enrichment ratio = 15.8) and the cellular response to cytokine stimulus (FDR = 3.1 x 10-9; enrichment ratio = 10.9) were highly enriched when mapped to these nodes. We conclude that CCN1, acting as a paracrine signal in the GBM tumor microenvironment, primes the cell to inhibit HSV-1 and other candidate OV replication prior to any virus interactions, thus contributing to immunovirotherapy resistance. While CCN1 and its cell surface integrin binding partners are ostensible drug targets for overcoming this resistance, pharmacologic interventions that target CCN1-specific high flow edges in our prioritized PPI network may be more effective. Citation Format: Dileep D. Monie, Cristina Correia, Cheng Zhang, Choong Ung, Richard G. Vile, Hu Li. Innate immunity and cytokine signaling pathways drive resistance to HSV-1 oncolytic immunovirotherapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1679.

Abstract 258: Identification of characteristic genes of scirrhous-type gastric cancer cells by RNAseq

Abstract Introduction: Scirrhous gastric cancer shows an extremely poor prognosis with rapid growth and infiltration activity. One of the reasons for poor prognosis is a lack of the identification of driver gene for scirrhous gastric cancer. FGFR2 (fibroblast growth factor receptor 2), E-cadherin, TGFβR (transforming growth factor β receptor), CD44/IGFIR (insulin-like growth factor I receptor), have been reported as candidates of driver for scirrhous-type of gastric cancer, however useful molecular-targeted therapeutic agents against these driver molecules have not yet been clinically approved. In this study, we identified the characteristic genes of scirrhous-type gastric cancer by RNA-seq of gastric cancer cell lines. Methods: RNA-seq was performed using 6 scirrhous gastric cancer cell lines (OCUM1, OCUM2MLN, OCUM8, OCUM12, OCUM13, OCUM14) using a next-generation sequencer (ION S5 system, Thermo Fisher Scientific). Furthermore, RNA-seq public data of 4 strains of scirrhous gastric cancer cell lines (KATO3, NUGC4, SNU601, SNU668) and 10 strains of non-scirrhous gastric cancer cell lines (MKN7, MKN45, MKN74, HS746T, SNU1, SNU16, SNU638, AZ521, HGC27, TMK1) on the Sequence Read Archive were added, and the genes expressed in scirrhous gastric cancer were compared and examined. The analysis tools used were kallisto and sleuth. We set p-value < 0.05 and log2 (fold change) > 2 as the cut-offs to screen out differentially expressed genes (DEGs). Pathway enrichment and protein-protein interaction (PPI) network analysis were performed based on metascape databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was also performed for the genes. Results: A total of 234 DEGs were identified from this analysis. A gene co-expression network was constructed containing 8 DEGs. The genes were mainly associated with response to chemokine, and cytokine-mediated signaling pathway. Several critical genes were disclosed, such as C-C motif chemokine ligand 20 (CCL20) and C-X-C subfamiliy. Genes highly expressed in scirrhous gastric cancer cell lines were CCL20 (10.6-fold change, p=9.89×10-4) and C-X-C Motif Chemokine Ligand 3 (CXCL3, 19.5-fold change, p=1.67×10-5). The mean of abundances across samples (=log(counts+0.5)), which represent the average expression levels of all samples, were CCL20 and CXCL3, 4.04 and 4.53, respectively. KEGG pathway enrichment analysis showed that viral protein interaction with cytokine and cytokine receptor, Cytokine-cytokine receptor interaction and IL-17 signaling pathway were significantly over-represented. Conclusion: CCL20 and CXCL3 might be characteristic genes for scirrhous-type of gastric cancer. Citation Format: Tomohiro Sera, Masakazu Yashiro, Gen Tsujio, Yurie Yamamoto, Atsushi Sugimoto, Shuhei Kushiyama, Sadaaki Nishimura, Masaichi Ohira. Identification of characteristic genes of scirrhous-type gastric cancer cells by RNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 258.

Zinc transporter SLC39A13/ZIP13 facilitates the metastasis of human ovarian cancer cells via activating Src/FAK signaling pathway

BackgroundZinc transporters have been found to be associated with the pathogenesis of numerous human diseases including cancer. As the most lethal gynecologic malignancy, ovarian cancer is characterized by rapid progression and widespread metastases. However, the function and underlying mechanism of zinc transporters in ovarian cancer metastasis remain unclear.MethodsThe relationship between zinc transporter gene expressions and clinical outcomes of ovarian cancer was assessed with the online database Kaplan-Meier plotter (http://kmplot.com/analysis/). Immunohistochemistry was performed to investigate the prognostic importance of ZIP13. The expression of ZIP13 in ovarian cancer cell lines was depleted to explore its effect on proliferation, adhesion, migration, and invasion both in vitro and in vivo assays. RNA-Seq, quantitative RT-PCR, and western blot analysis were performed to explore ZIP13-regulated downstream target genes.ResultsThe expressions of several zinc transporters were highly associated the clinical outcomes of ovarian cancer patients. Among them, high ZIP13 expression was an independent prognostic factor for poor survival in patients with ovarian cancer. ZIP13 knockout suppressed the malignant phenotypes of ovarian cancer cells both in vitro and in vivo. Further investigation revealed that ZIP13 regulated intracellular zinc distribution and then affected the expressions of genes involved in extracellular matrix organization and cytokine-mediated signaling pathway. This led to the activation of Src/FAK pathway with increased expressions of pro-metastatic genes but decreased expressions of tumor suppressor genes.ConclusionsZIP13 is shown to be a novel driver of metastatic progression by modulating the Src/FAK signaling pathway, which may serve as a promising biomarker for prognostic evaluation and targeted therapy in ovarian cancer.

Genome-Wide Differential Expression Profiling of Pulmonary circRNAs Associated With Immune Reaction to Pasteurella multocida in Goats

Pasteurella multocida is a highly versatile pathogen that infects a wide range of animals, including goats, causing pneumonia and hemorrhagic septicemia. Circular RNA (circRNA) is a type of non-coding RNA that plays an important role in regulating cellular metabolism. However, whether and how circRNA is involved in regulating immune responses in the goat lung has not been reported. Thus, this study was designed to examine the function of circRNA in goats infected with Pasteurella multocida. Goats were assigned into one of two groups: an uninfected control group (CK) and an infected group challenged with P. multocida. Compared with the CK group, which remained healthy, the infected goats showed clinical signs of infection, including depression, cough, nasal discharge, and dyspnea, along with elevated body temperature and lesions in the lung. Whole-transcriptome sequencing and small RNA sequencing were then performed using lung samples from goats from each group. A total of 138 circRNA, 56 microRNAs (miRNA), and 2,673 messenger RNA (mRNA) molecules were significantly differentially expressed in the P. multocida-infected group compared with the CK group. Randomly selected differentially expressed circRNA, miRNA, and mRNA molecules (n = 5 per group) were then validated by quantitative reverse-transcriptase polymerase chain reaction analysis. Gene ontology (GO) analysis of the source genes indicated that six immune-related terms were enriched among the differentially expressed cirRNA molecules, including inflammatory response, immune effector process, cell activation involved in immune response, cytokine-mediated signaling pathway, response to endogenous stimulus, and immune response. The corresponding circRNA molecules were then selected for construction of a competitive endogenous RNA network to identify networks that may be involved in the immune response to P. multocida infection. The results indicated that P. multocida HN01 may cause pneumonia and stimulate an immune response in goats via regulation of circRNA expression. This study presents the first comprehensive circRNA profile in response to P. multocida infection in goats, thus, providing a basis for understanding the function of circRNA in the host immune response to P. multocida infection.

Investigation of the Mechanisms of Chuankezhi Injection in the Treatment of Asthma Based on the Network Pharmacology Approach.

Background Chuankezhi injection (CKZI) was an effective traditional Chinese medicine (TCM) injection in adjuvant bronchial asthma therapy. In this report, we used a network pharmacology method to reveal the mechanisms of CKZI for the treatment of asthma. Methods The candidate compounds in CKZI were determined by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and China National Knowledge Infrastructure website (CNKI). The targets of candidate compounds were searched in the TCMSP, DrugBank 5.0, and SwissTargetPrediction. The disease targets were screened from the Online Mendelian Inheritance in Man (OMIM) and GeneCards. The overlapping gene symbols between candidate compounds and disease were filtered via a Venn diagram and were considered as potential targets. A protein-protein interaction (PPI) network and disease-related candidate compound-target-pathway (DC-T-P) network were visualized by Cytoscape 3.6.1. Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by metascape to determine the pathways related to asthma. Results A total of 70 overlapping gene symbols were recognized as potential targets. Cytokines (IL6, TNF, and IL1B) and chemokines (CXCL8 and CCL2) could be recognized as hub genes. Asthma-related candidate compounds were mainly flavonoids, such as quercetin, luteolin, and kaempferol. The cytokine-mediated signaling pathway, cytokine receptor binding, and membrane craft were the most significant biological process (BP), molecular function (MF), and cellular component (CC) of GO function results, respectively. The relevant pathways of CKZI against asthma mainly include IL-17, NF-kappa B, HIF-1, calcium, and PI3K-Akt signaling pathways. Conclusion Our research provided a theoretical basis for further investigating the mechanisms of CKZI in the treatment of asthma.

Molecular Targets and Mechanisms of Scutellariae radix-Coptidis rhizoma Drug Pair for the Treatment of Ulcerative Colitis Based on Network Pharmacology and Molecular Docking.

This study aims to analyze the targets of the effective active ingredients of Scutellariae radix-Coptidis rhizoma drug pair (SCDP) in ulcerative colitis (UC) by network pharmacology and molecular docking and to explore the associated therapeutic mechanism. The effective active ingredients and targets of SCDP were determined from the TCMSP database, and the drug ingredient-target network was constructed using the Cytoscape software. The disease targets related to UC were searched in GeneCards, DisGeNET, OMIM, and DrugBank databases. Then, the drug ingredient and disease targets were intersected to construct a protein-protein interaction network through the STRING database. The Metascape database was used for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the predicted targets of SCDP for UC. The Autodock software was used for molecular docking between the main active ingredient and the core target to evaluate the binding ability. SCDP has 43 effective active ingredients and 134 intersection targets. Core targets included AKT1, TP53, IL-6, VEGFA, CASP3, JUN, TNF, MYC, EGFR, and PTGS2. GO functional enrichment analysis showed that biological process was mainly associated with a cytokine-mediated signaling pathway, response to an inorganic substance, response to a toxic substance, response to lipopolysaccharide, reactive oxygen species metabolic process, positive regulation of cell death, apoptotic signaling pathway, and response to wounding. KEGG enrichment analysis showed main pathway concentrations were related to pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, bladder cancer, IL-17 signaling pathway, apoptosis, p53 signaling pathway, and PI3K-Akt signaling pathway. The drug active ingredient-core target-key pathway network contains 41 nodes and 108 edges, of which quercetin, wogonin, baicalein, acacetin, oroxylin A, and beta-sitosterol are important active ingredients; PTGS2, CASP3, TP53, IL-6, TNF, and AKT1 are important targets; and the pathways involved in UC treatment include pathways in cancer, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic, apoptosis, IL-17 signaling pathway and herpes simplex infection. The active ingredient has a good binding capacity to the core target. SCDP key active ingredients are mainly quercetin, wogonin, baicalein, acacetin, oroxylin A, and beta-sitosterol, which function mainly by regulating targets, such as PTGS2, CASP3, TP53, IL-6, TNF, and AKT1, and are associated with multiple signaling pathways as pathways in cancer, PI3K-Akt signaling pathway, apoptosis, IL-17 signaling pathways.