PCR Array Analysis Research Articles

Interleukin-35 expression promotes hepatocellular carcinogenesis by inducing γδ T-cell exhaustion

Interleukin (IL)-35, a new member of the IL-12 family, exerts immunosuppressive effects in the hepatic microenvironment. Innate immune cells, such as γδ T cells, have vital roles in hepatic diseases, including acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). In the current study, we focused on the effects and mechanisms of IL-35 on the local immune status of γδ T cells, especially in liver tumors. Based on CCK8 assay and immunofluorescence results, we showed that exogenous IL-35 stimulation of γδ T cells attenuated proliferative ability and killing functions against Hepa1–6 or H22 cells. Flow cytometry results showed that exogenous IL-35 stimulated the expression of programmed cell death 1 (PDCD1) and lymphocyte activation gene 3 (LAG3) in γδ T cells. The exogenous IL-35 stimulated group also showed impairment of cytotoxic cytokine secretion. In addition, stat5a revealed a significant increase after IL-35 stimulation of γδ T cells screened via transcription factor based on PCR array analysis. Furthermore, bioinformatics analysis revealed that stat5a-related tumor-specific genes were mainly involved in immune regulatory pathways. Correlation analysis indicated that stat5a expression was significantly and positively correlated with tumor immune cell infiltration, and pdcd1 and lag3 expression. Finally, bioinformatics analysis using the TCGA and GSE36376 HCC datasets corroborated the significant positive correlation between IL-35 and stat5a. Taken together, overexpressed IL-35 promoted exhaustion and impaired the anti-tumor function of γδ T cells in HCC. Targeting IL-35 might be a promising means to enhance the antitumor therapy of γδ T cells, which would significantly improve prognosis.

β-defensin-1 regulates influenza virus infection in human bronchial epithelial cells through the STAT3 signaling Pathway

Abstract To develop intervention strategies it is necessary to understand the host response to influenza A virus (IAV) infection. The primary barriers for invading respiratory pathogens are the airway epithelial cells of the respiratory tract and several antimicrobial peptides are produced by these cells. The antimicrobial peptide, β-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of β-defensin-1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis, plaque assay, and confocal microscopy. DEFB1expression after transfection with two micro RNAs (miRNAs), hsa-miR-186-5p and hsa-miR-340-5p, provided evidence that DEFB1expression could be modulated by these miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1in IAV infected HBEpCs led to increased NF-κB expression. In a PCR array analysis of 84 transcription factors, either overexpressing DEFB1or siRNA silencing of DEFB1expression significantly modulated the expression of signal transducer and activator of transcription 3 (STAT3). In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered in cells overexpressing DEFB1, suggesting this to be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1overexpressing cells suggests that β-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development. No funding

Restoration of coronary microvascular function by OGA overexpression in a high-fat diet with low-dose streptozotocin-induced type 2 diabetic mice.

Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

Molecular Mechanisms of the Anti-Inflammatory Effects of Epigallocatechin 3-Gallate (EGCG) in LPS-Activated BV-2 Microglia Cells.

Chronic neuroinflammation is associated with many neurodegenerative diseases, such as Alzheimer's. Microglia are the brain's primary immune cells, and when activated, they release various proinflammatory cytokines. Several natural compounds with anti-inflammatory and antioxidant properties, such as epigallocatechin 3-gallate (EGCG), may provide a promising strategy for inflammation-related neurodegenerative diseases involving activated microglia cells. The objective of the current study was to examine the molecular targets underlying the anti-inflammatory effects of EGCG in activated microglia cells. BV-2 microglia cells were grown, stimulated, and treated with EGCG. Cytotoxicity and nitric oxide (NO) production were evaluated. Immunoassay, PCR array, and WES™ Technology were utilized to evaluate inflammatory, neuroprotective modulators as well as signaling pathways involved in the mechanistic action of neuroinflammation. Our findings showed that EGCG significantly inhibited proinflammatory mediator NO production in LPS-stimulated BV-2 microglia cells. In addition, ELISA analysis revealed that EGCG significantly decreases the release of proinflammatory cytokine IL-6 while it increases the release of TNF-α. PCR array analysis showed that EGCG downregulated MIF, CCL-2, and CSF2. It also upregulated IL-3, IL-11, and TNFS10. Furthermore, the analysis of inflammatory signaling pathways showed that EGCG significantly downregulated mRNA expression of mTOR, NF-κB2, STAT1, Akt3, CCL5, and SMAD3 while significantly upregulating the expression of mRNA of Ins2, Pld2, A20/TNFAIP3, and GAB1. Additionally, EGCG reduced the relative protein expression of NF-κB2, mTOR, and Akt3. These findings suggest that EGCG may be used for its anti-inflammatory effects to prevent neurodegenerative diseases.

The impact of cellular senescence and senescence‑associated secretory phenotype in cancer‑associated fibroblasts on the malignancy of pancreatic cancer.

Cancer‑associated fibroblasts (CAFs) are implicated in the strong malignancy of pancreatic cancer (PC). Various CAF subtypes have different functions, and their heterogeneity likely influence the malignancy of PC. Meanwhile, it is known that senescent cells can create a tumor‑promoting microenvironment by inducing a senescence‑associated secretory phenotype (SASP). In the present study, the effects of individual differences in CAFs on PC malignancy were investigated with a focus on cellular senescence. First, primary cultures of CAFs from 8 PC patients were generated and co‑cultured with PC cell lines. This co‑culture assay showed that differences in CAFs induce differences in PC cell proliferation. It was further investigated which clinical factors affected the malignant potential of CAF and it was found that the difference of malignant potential of each CAF was marginally related to the age of original patients. Next, to verify the senescence of CAFs really affected the malignant potential of CAF, PCR array analysis of each CAF sample was performed and it was revealed that expression of genes about cellular senescence and SASP such as tumor protein p53, nuclear factor kappa B subunit 1, and IL6, are related to the malignant potential of CAFs impacting on PC proliferation. Finally, to elucidate the effect of p53‑mediated cellular senescence of CAFs on malignant potential of PC, it was examined whether CAFs with the treatment of p53 inhibitor affected PC cell proliferation in co‑culture assays. The treatment of CAFs with p53 inhibitor significantly suppressed PC cell proliferation. In addition, a comparison of the concentration of IL‑6, a SASP cytokine, in the co‑culture supernatant showed a significant decrease in the sample after p53 inhibitor treatment. In conclusion, the present results suggested that proliferation potential of PC may be related to p53‑mediated cellular senescence and SASP of CAFs.

MicroRNAs as Biomarkers of Active Pulmonary TB Course

The spread of drug-resistant forms of TB dictates the need for surgical treatment in the complex of anti-tuberculosis measures in Russia. Most often, surgical intervention is performed in the case of pulmonary tuberculoma or fibrotic cavitary tuberculosis (FCT). This study is devoted to the search for biomarkers that characterize the course of disease in surgical TB patients. It is assumed that such biomarkers will help the surgeon decide on the timing of the planned operation. A number of serum microRNAs, potential regulators of inflammation and fibrosis in TB, selected on the basis of PCR-Array analysis, were considered as biomarkers. Quantitative real time polymerase chain reaction and receiver operating curves (ROC) were used to verify Array data and to estimate the ability of microRNAs (miRNAs) to discriminate between healthy controls, tuberculoma patients, and FCT patients. The study showed that miR-155, miR-191 and miR-223 were differentially expressed in serum of tuberculoma with "decay" and tuberculoma without "decay" patients. Another combination (miR-26a, miR-191, miR-222 and miR-320) forms a set to differentiate between tuberculoma with "decay" and FCT. Patients with tuberculoma without "decay" diagnosis differ from those with FCT in serum expression of miR-26a, miR-155, miR-191, miR-222 and miR-223. Further investigations are required to evaluate these sets on a larger population so as to set cut-off values that could be applied in laboratory diagnosis.

β-Defensin-1 Regulates Influenza Virus Infection in Human Bronchial Epithelial Cells through the STAT3 Signaling Pathway.

Understanding the host response to influenza A virus (IAV) infection is vital for developing intervention strategies. The primary barriers for invading respiratory pathogens are the respiratory tract epithelial cells and antimicrobial proteins generated by these cells. The antimicrobial peptide, β-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of β-defensin1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1 caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis, plaque assay, and confocal microscopy. DEFB1 expression after transfection with two micro RNAs (miRNAs), hsa-miR-186-5p and hsa-miR-340-5p, provided evidence that DEFB1 expression could be modulated by these miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1 in IAV-infected HBEpCs led to increased NF-κB expression. In a PCR array analysis of 84 transcription factors, either overexpressing DEFB1 or siRNA silencing of DEFB1 expression significantly modulated the expression of signal transducer and activator of transcription 3 (STAT3). In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered in cells overexpressing DEFB1, suggesting this to be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1 overexpressing cells suggests that β-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms.

Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

Paeoniae Radix Rubra can enhance fatty acid β-oxidation and alleviate gut microbiota disorder in α-naphthyl isothiocyanate induced cholestatic model rats.

Cholestasis is the most destructive pathological manifestation of liver disease and available treatments are very limited. Paeoniae Radix Rubra (PRR) is an important traditional Chinese drug used to treat cholestasis. This study combined targeted metabonomics, PCR array analysis, and 16S rRNA sequencing analysis to further clarify the mechanisms of PRR in the treatment of cholestasis. PRR conspicuously reversed the elevation of fatty acids (FFA 14:0 and other 14 fatty acids) and the decrease of organic acids (pyruvic acid and citric acid) in a cholestatic model induced by α-naphthyl isothiocyanate (ANIT). Eight elevated amino acids (L-proline, etc.) and five elevated secondary bile acids (taurohyodeoxycholic acid, etc.) in model rats were also reduced by PRR. Pathway analysis revealed that PRR significantly alleviated eight pathways (β-alanine metabolism). Furthermore, we found that PRR significantly reversed the decrease of Cpt1a, Hadha, Ppara, and Slc25a20 (four genes relevant to fatty acid β-oxidation) mRNAs caused by ANIT, and PRR conspicuously decreased nine acylcarnitines (the forms of fatty acids into mitochondria for β-oxidation) that increased in model rats. These results indicate that PRR could enhance fatty acid β-oxidation, which may be the way for PRR to reduce the levels of 15 fatty acids in the serum of model rats. 16S rRNA sequencing analysis revealed that PRR alleviated gut microbiota disorders in model rats, including upregulating four genera (Coprococcus, Lactobacillus, etc.) and downregulating four genera (Bacteroides, Escherichia, etc.). As the relative abundance of these eight genera was significantly correlated with the levels of the five secondary bile acids (deoxycholic acid, taurolithocholic acid, etc.) reduced by PRR, and Bacteroides and Escherichia were reported to promote the production of secondary bile acid, we inferred that the downregulation of PRR on five secondary bile acids in model rats was inseparable from gut microbiota. Thus, the gut microbiota also might be a potential pharmacological target for the anticholestatic activity of PRR. In conclusion, we consider that the mechanisms of PRR in treating cholestasis include enhancing fatty acid β-oxidation and alleviating gut microbiota disorders.

Identification of oncogenic signatures in the inflammatory colon of C57BL/6 mice fed a high-fat diet

Adoption of an obesogenic diet such as a high-fat diet (HFD) results in obesity, bacterial dysbiosis, chronic inflammation, and cancer. Gut bacteria and their metabolites are recognized by interleukin-1 (IL-1R)/toll-like receptors (TLRs) which are essential to maintain intestinal homeostasis. Moreover, host extracellular microRNAs (miRNAs) can alter bacterial growth in the colon. Characterization of the underlying mechanisms may lead to identifying fecal oncogenic signatures reflecting colonic health. We hypothesize that an HFD accelerates the inflammatory process and modulates IL-1R/TLR pathways, gut microbiome, and disease-related miRNA in the colon. In this study, 4-week-old C57BL/6 mice were fed a modified AIN93G diet (AIN, 16% energy fat) or an HFD (45% energy fat) for 15 weeks. In addition to increased body weight and body fat composition, the concentrations of plasma interleukin 6 (IL-6), inflammatory cell infiltration, β-catenin, and cell proliferation marker (Ki67) in the colon were elevated > 68% in the HFD group compared to the AIN group. Using a PCR array analysis, we identified 14 out of 84 genes with a ≥ 24% decrease in mRNA content related to IL-1R and TLR pathways in colonic epithelial cells in mice fed an HFD compared to the AIN. Furthermore, the content of Alistipes bacteria, the Firmicutes/Bacteroidetes ratio, microRNA-29a, and deoxycholic and lithocholic acids (secondary bile acids with oncogenic potential) were 55% greater in the feces of the HFD group compared to the AIN group. Collectively, this composite, a multimodal profile may represent a unique HFD-induced fecal signature for colonic inflammation and cancer in C57BL/6 mice.

Transcriptomic Analysis of Genes Associated with Oxidative Stress in Chronic Rhinosinusitis Patients with Nasal Polyps: Identifying Novel Genes Involved in Nasal Polyposis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complicated inflammatory disease, and the underlying mechanism remains unclear. While some reactive oxygen/nitrogen species-related gene products are reported to participate in CRSwNP, a systemic and full analysis of oxidative-stress-associated genes in CRSwNP has not been extensively studied. Therefore, this study sought to catalog the gene-expression patterns related to oxidative stress and antioxidant defense in control and CRSwNP patients. In total, 25 control and 25 CRSwNP patients were recruited. The distribution and expression of 4-hydroxynonenal and 3-nitrotyrosine as markers of oxidative stress—which is represented by lipid peroxidation and the protein nitration of tyrosine residues in CRSwNP nasal polyps (NPs)—were more apparently increased than those found in the control nasal mucosae, as determined by immunohistochemistry (IHC). The expression of 84 oxidative-stress-related genes in nasal mucosae and NP tissues was analyzed via real-time PCR, which showed that 19 genes and 4 genes were significantly up- and downregulated, respectively; among them, inducible nitric oxide synthase (iNOS) and heme oxygenase 1 (HO-1) were notably upregulated, whereas lactoperoxidase (LPO), myeloperoxidase (MPO), and superoxide dismutase 3 (SOD3) were highly downregulated. Changes in the mRNA and protein levels of these redox proteins were confirmed with a customized, real-time PCR array and RT-PCR analysis, as well as Western blotting and IHC assays. A receiver operating characteristic curve analysis further suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors of CRSwNP development. Collectively, we present an oxidative-stress-related gene profile of CRSwNP NP tissues, providing evidence that the systemic changes in oxidative stress and the antioxidative defense system, including novel iNOS, heme peroxidases, and other genes, are closely linked to CRSwNP pathology, development, and progression.

IL-9 neutralizing antibody suppresses allergic inflammation in ovalbumin-induced allergic rhinitis mouse model.

Allergic rhinitis is mainly mediated by IgE after specific individuals are exposed to allergens. It is a common nasal mucosa disease of non-infectious chronic inflammatory disease and is often accompanied by asthma and conjunctivitis. In the study of allergic asthma, it was found that IL-9 participates in the pathogenic development of asthma. Because asthma and allergic rhinitis have the same airway and the same disease, it is inferred that IL-9 may also play an important role in allergic rhinitis. BALB/c mice received intranasal stimulation of ovalbumin (OVA) treatment at different times. The nasal mucosa of the mice were then sliced and stained with Sirius red and Toluidine blue, and eosinophils and mast cells in the mucosa were counted. ELISA was used to detect the expression of OVA-IgE in peripheral blood. The Th2 cell fraction in the mouse spleen was detected by flow cytometry. The expressions of IL-4, IL-5, IL-9, and IL-13 and their mRNA in mucosa were detected by real-time PCR and flow cytometry bead array analysis. Finally, the expression changes of Thymic stromal lymphopoietin related proteins and its mRNA, JAK1/2, and STAT5 proteins were detected by real-time PCR and Western blot. After the intervention with the IL-9 neutralizing antibody, the symptoms of allergic rhinitis in mice were significantly reduced. The expression of OVA-IgE in the peripheral blood of mice was inhibited, the fraction of Th2 cells in the spleen decreased, the related cytokines (IL-4, IL-5, and IL-13) were inhibited, and their functions decreased. The TSLP-OX40/OX40L signal pathway and JAK1/2-STAT5 signal are inhibited. IL-9 neutralizing antibody has a good therapeutic effect on the mouse model of allergic rhinitis, which may be related to the TSLP-OX40/OX40L pathway and JAK1/2-STAT5 signaling.

Biochemical, pharmacological, and toxicological attributes of caper (Capparis ovata) flowering buds and berries pickles.

Capparis ovata is a natural plant that grows widely in Turkey and its flowering buds and berry pickle are used in traditional medicine. Thus, the current study was expanded to evaluate the biochemical, pharmacological, and toxicological aspects of the Capparis ovata water extract (COWE). To determine the biochemical properties of COWE, mineral and fatty acid content, elemental analysis, flavonoid/phenolic content, radical-scavenging capacity, and pesticide analysis were performed. Furthermore, to find out whether it had anti-inflammatory properties, reverse transcription-polymerase chain reaction (RT-PCR) and nuclear factor kappa B (NF-κB) luciferase activity tests were conducted. Whole-genome transcriptomic profiling was carried out at a dose level of 500 mg/kg COWE to understand its pharmacological effect. Transaminases in serum were tested, and quantitative polymerase chain reaction (qPCR) was done using a custom design array that included the stress and molecular toxicology pathway to establish its toxicological qualities. As a result of the evaluations, it was observed that COWE has a high mineral and unsaturated fatty acid content, flavonoid/phenolic content, and radical-scavenging ability. It significantly inhibited NF-κB transcriptional activity as well as inflammatory cytokine expression in T-lymphoblast cells. Whole-genome transcriptomic profiling depicted that COWE modulates immune responses by upregulating natural killer cell activation, cellular response to type I interferon, B-cell proliferation and differentiation, and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Molecular Toxicology Pathfinder RT2 Profiler PCR array analysis revealed that COWE at or lower dose of 500 mg/kg/day did not cause a comparatively adverse effect. According to the findings, COWE is a rich source of nutrients and can be used as an adjunct therapy for various inflammatory diseases.

Multiple exposure to methylmercury aggravates DNA damage in the BTBR T + Itpr3 tf/J autistic mouse model: The role of DNA repair efficiency

Environmental and genetic factors have been recognized to play major roles in the pathogenesis of autism. Here we examined the BTBR T+Itpr3tf/J (BTBR) mice’s susceptibility, an autistic model, to the genotoxic effects and DNA repair dysregulation of methylmercury. Micronuclei formation and oxidative DNA damage were analyzed using the micronucleus/fluorescence in situ hybridization test and modified comet assay, respectively. The results showed higher centromeric-positive micronuclei and oxidative DNA damage in BTBR mice exposed to methylmercury than the unexposed mice, which indicates that mutagenesis aggravated in BTBR mice after methylmercury exposure. Lipid peroxides in BTBR mice were significantly elevated, with a decrease in reduced/oxidized glutathione ratio after methylmercury exposure, indicating an augmenting oxidant-antioxidant imbalance. The expression of several genes involved in DNA repair was markedly altered in BTBR mice after methylmercury exposure as evaluated via PCR array and RT-PCR analyses. Declining of the antioxidant defense and dysregulation in DNA repair process after methylmercury exposure may explain the aggravated genotoxic susceptibility of BTBR mice. Thus, autistic individuals exposed to methylmercury must be under regular medical follow-up through standard timetabled medical laboratory inquiry to allow for early recognition of any mutagenic changes. Additionally, strategies that elevate cellular antioxidants/DNA repair efficiency may counteract methylmercury-induced genotoxicity.

Abstract 1477: Circulating miR-125a-3p and miR-451a may be liquid biopsy biomarkers for the diagnosis of breast cancer

Abstract Background: Breast cancer is the most prevalent female cancer. Emerging evidence suggests that the unique serum miRNAs can assist in the early detection and diagnosis of breast cancer. Cell-free circulating miRNAs (about 22 nucleotides) are potential diagnostic tools and minimally invasive biomarkers for breast cancer detection. This study aimed to identify specific microRNAs in the serum of patients with breast cancer, which may serve as promising diagnostic and prognostic biomarkers and therapeutic targets. Material and methods: Next-generation sequencing and quantitative real-time PCR (qRT-PCR) array analyses of microRNAs in sera from different subtypes of breast cancer patients and normal female individuals were performed. The differentially expressed microRNAs were verified in serum samples from 100 breast cancer patients. The predictive values of the selected microRNAs were statistically analyzed, determined by the correlation between microRNA expression and tumor parameters. Datasets were graphed using GraphPad Prism Software 8.0. and analysis was done using one-way ANOVA with unpaired two-tailed Student’s t-test. Result: High serum levels of miR-125a-3p and miR-451a were found to be associated with breast cancer tumor subtype, size, and lymph node metastasis. Further functional in vitro experiments and enrichment analysis showed that overexpression and knockdown of miR-451 might significantly affect tumorigenic signaling pathways in TNBC cells. Conclusion: Altogether, our data suggested that serum levels of microRNAs miR-125a-3p and miR-451a can potentially serve as liquid biopsy biomarkers in breast cancer. Citation Format: Hassan Yousefi, Steven C. Eastlack, Jordyn T Fong, Samuel C. Okpechi, Jovanny Zabaleta, Lyndsey R. Buckner, Donnalee N Trapani, Erin Sims, Alison McDaniel McDaniel, Heather Scuderi, Meredith A. Lakey, John T. Cole, Suresh K. Alahari. Circulating miR-125a-3p and miR-451a may be liquid biopsy biomarkers for the diagnosis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1477.

Identification of Small Nucleolar RNA SNORD60 as a Potential Biomarker and Its Clinical Significance in Lung Adenocarcinoma.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer deaths in the world and often diagnosed at an advanced stage, so it is urgent to explore the pathogenesis and new diagnostic biomarkers. Accumulated evidences suggested that small nucleolar RNAs (snoRNAs) played a key role in the development and progression of NSCLC. To examine differential expression snoRNA profile and identify snoRNAs with clinical significance in lung adenocarcinoma (LUAD), The Cancer Genome Atlas (TCGA) LUAD RNA sequencing dataset was used to investigate differential expression snoRNA signatures and compared with snoRNA PCR array analysis in pair-matched LUAD tissues. The diagnostic ability of SONRD60 was assessed using a receiver operating characteristic (ROC) curve. The Kaplan-Meier method was used to plot survival curves. Univariate and multivariate Cox regression analyses were used to investigate the prognostic effect of SNORD60 expression on LUAD. The results showed that SNORD60 was a significantly upregulated snoRNA after intersection analysis in LUAD cases. SNORD60 has 74.2% sensitivity and 75.3% specificity for the diagnosis of LUAD. Increased SNORD60 expression was linked with lymph node metastases and the TNM stage (P < 0.05). Pathological T category and lymph node metastases were independent prognostic factors for overall survival in a multivariate Cox regression study. Our findings demonstrated that SNORD60, a small nucleolar RNA, has an oncogenic function in LUAD and might be used as a new early diagnostic biomarker for LUAD.

Mechanistic insights gained from cell and molecular analysis of the neuroprotective potential of bioactive natural compounds in an immortalized hippocampal cell line.

Evaluating novel compounds for neuroprotective effects in animal models of traumatic brain injury (TBI) is a protracted, labor-intensive and costly effort. However, the present lack of effective treatment options for TBI, despite decades of research, shows the critical need for alternative methods for screening new drug candidates with neuroprotective properties. Because natural products have been a leading source of new therapeutic agents for human diseases, we used an in vitro model of stretch injury to rapidly assess pro-survival effects of three bioactive compounds, two isolated from natural products (clovanemagnolol [CM], vinaxanthone [VX]) and the third, a dietary compound (pterostilbene [PT]) found in blueberries. The stretch injury experiments were not used to validate drug efficacy in a comprehensive manner but used primarily, as proof-of-principle, to demonstrate that the neuroprotective potential of each bioactive agent can be quickly assessed in an immortalized hippocampal cell line in lieu of comprehensive testing in animal models of TBI. To gain mechanistic insights into potential molecular mechanisms of neuroprotective effects, we performed a pathway-specific PCR array analysis of the effects of CM on the rat hippocampus and microRNA sequencing analysis of the effects of VX and PT on cultured hippocampal progenitor neurons. We show that the neuroprotective properties of these natural compounds are associated with altered expression of several genes or microRNAs that have functional roles in neurodegeneration or cell survival. Our approach could help in quickly assessing multiple natural products for neuroprotective properties and expedite the process of new drug discovery for TBI therapeutics.

Analgesic Effect of Tranilast in an Animal Model of Neuropathic Pain and Its Role in the Regulation of Tetrahydrobiopterin Synthesis.

Trigeminal neuralgia is unilateral, lancinating, episodic pain that can be provoked by routine activities. Anticonvulsants, such as carbamazepine, are the drugs of choice; however, these possess side-effects. Microvascular decompression is the most effective surgical technique with a higher success rate, although occasionally causes adverse effects. The potential treatment for this type of pain remains unmet. Increased tetrahydrobiopterin (BH4) levels have been reported in association with axonal injury. This study aimed to evaluate the effect of tranilast on relieving neuropathic pain in animal models and analyze the changes in BH4 synthesis. Neuropathic pain was induced via infraorbital nerve constriction. Tranilast, carbamazepine, or saline was injected intraperitoneally to assess the rat’s post-intervention pain response. In the von Frey’s test, the tranilast and carbamazepine groups showed significant changes in the head withdrawal threshold in the ipsilateral whisker pad area. The motor coordination test showed no changes in the tranilast group, whereas the carbamazepine group showed decreased performance, indicating impaired motor coordination. Trigeminal ganglion tissues were used for the PCR array analysis of genes that regulate the BH4 pathway. Downregulation of the sepiapterin reductase (Spr) and aldoketo reductase (Akr) genes after tranilast injection was observed compared to the pain model. These findings suggest that tranilast effectively treats neuropathic pain.

Diet Significantly Influences the Interleukin‐6 Response to Mouse Models of Peritoneal Sepsis

IntroductionSepsis remains to be a tremendous health burden in the United States, affecting 1.7 million Americans and causing over 250,000 deaths each year. Previous work has shown high‐calorie diets to significantly alter the immune response and outcomes in animal models of sepsis; however, the diets used model overt metabolic dysfunction and not dietary patterns observed in a “typical” American. We developed a novel Americanized diet (AD) that incorporates several of the nutritional inadequacies reported in Americans and set out to determine how consumption of the AD influences immunological outcomes of peritoneal sepsis in mice.MethodologyMale C57Bl/6 mice were used in accordance to protocols approved by the Institutional Animal Care and Use Committee at LUCOM and compliant with the Guide for the Care and Use of Laboratory Animals. Mice received ad libitum access to experimental diets [chow, AD, or a commercially available high‐calorie Western diet (WD)] for 8‐weeks and peritonitis was induced with cecal‐ligation and puncture (CLP) or intraperitoneal injection of a fecal slurry (fecal peritonitis, “FP”). For both CLP and FP, blood was collected 24‐hours later for quantification of circulating interleukin‐6 (IL6). Another group of mice were fed their assigned diets and the spleens cultured ex vivo in DMEM media with lipopolysaccharide (LPS, 10ug/mL) or saline for 3 hours. Tissue and supernatant were collected and frozen until assayed for IL6 and inflammatory gene expression using a commercially available PCR‐microarray. All data were analyzed using general linear model procedures in SPSS with Tukey’s post hoc test and group averages were considered statistically significant with a P‐value less than 0.05.ResultsMice consuming AD and WD had ~3‐fold greater circulating IL6 following CLP (P=0.04) and FP (P&lt;0.05) as compared to mice consuming chow. LPS stimulation of splenic tissue ex vivo caused a significant increase in secreted IL6 protein (P&lt;0.001). Mice fed WD had the greatest IL6 concentration (425 pg/mL), which was greater than mice fed chow (163 pg/mL, P=0.04) but not mice fed AD (360 pg/mL, P=0.9). PCR‐array analysis confirmed the upregulation of IL6, with mice fed WD having double (P=0.03) the mRNA expression of splenic IL6 after LPS as compared to mice fed AD and chow. CCL12 expression was similarly upregulated in mice fed WD as compared to mice fed AD and chow (P=0.005).ConclusionOur data support the hypothesis that diet significantly influences the severity of animal models of sepsis. Consumption of a nutritionally inadequate AD caused a similar increase in circulating IL6 as compared to mice consuming a high calorie WD. However, the source of the additional circulating IL6 is unknown, ex vivo spleen stimulation studies with LPS did not report an increase in IL6 production in mice fed the AD. Future studies will determine if and how diet influences IL6 production in other tissues.

Evaluation of antitumor effects of VEGFR-2 inhibitor F16in a colorectal xenograft model.

Colorectal cancer(CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5μM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 valueof 9.52 ± 1.49µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showeda reduction in CD31 levels also in F16 treated groups. These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.