Quantitative Real-time Reverse Transcriptase PCR Research Articles

Abstract 3201: Complement component C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and its incidence is increasing globally. We have examined the role of complement component C3 and complement factor B (CFB) in the progression of cSCC. Significantly elevated mRNA levels for C3 and CFB were detected din cSCC cell lines (n = 8) compared to normal human epidermal keratinocytes (n = 11) by quantitative real-time reverse transcriptase-PCR. Increased production of C3 and CFB by cSCC cell lines was detected by western blotting. The mRNA levels for C3 and CFB were also markedly higher in cSCC tumors (n = 6) than in normal skin (n = 10). Tumor cell specific staining for C3 and CFB was detected in cSCC tumors in vivo by immunohistochemistry and the staining intensity was stronger in invasive cSCCs (n = 71) than in cSCC in situ (n = 69), premalignant epidermal lesions (actinic keratoses, n = 65) and normal skin (n = 5). Significant upregulation of C3 and CFB mRNA expression was also noted in chemically induced mouse skin cSCCs (n = 27) compared to benign papillomas (n = 17). The expression of C3 and CFB at mRNA and protein level was markedly higher in invasive tumorigenic Ha-ras-transformed HaCaT cell line than in benign tumorigenic ras-transformed HaCaT cells and parental non-tumorigenic HaCaT cells. The expression of both C3 and CFB was significantly upregulated by IFN-γ and TNF-α in cSCC cells. Knockdown of CFB and C3 by specific siRNAs inhibited migration of cSCC cells. Knockdown of CFB inhibited proliferation of cSCC cells and this was associated with potent inhibition of ERK1/2 activation. Knockdown of C3 and CFB also significantly inhibited growth of human cSCC xenograft tumors in vivo in SCID mice. These results provide evidence for the role of C3 and CFB in progression of cSCC and identify them as specific biomarkers and putative therapeutic targets for invasive cSCC. Citation Format: Pilvi Riihila, Mehdi Farshchian, Markku Kallajoki, Atte Kivisaari, Seppo Meri, Reidar Grenman, Ritva Heljasvaara, Taina Pihlajaniemi, Juha Peltonen, Sirkku Peltonen, Veli-Matti Kahari. Complement component C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3201. doi:10.1158/1538-7445.AM2015-3201

A Variegated Squirrel Bornavirus Associated with Fatal Human Encephalitis

Between 2011 and 2013, three breeders of variegated squirrels (Sciurus variegatoides) had encephalitis with similar clinical signs and died 2 to 4 months after onset of the clinical symptoms. With the use of a metagenomic approach that incorporated next-generation sequencing and real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), the presence of a previously unknown bornavirus was detected in a contact squirrel and in brain samples from the three patients. Phylogenetic analyses showed that this virus, tentatively named variegated squirrel 1 bornavirus (VSBV-1), forms a lineage separate from that of the known bornavirus species. (Funded by the Federal Ministry of Food and Agriculture [Germany] and others.)

Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1).

Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH), which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR), semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1) and liver X receptor (LXR)-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO) staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL) and with or without CRH (10 nM) in the presence of apolipoprotein A1 (apoA1) revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY)-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473) induced by interaction between CRH and CRH receptor 1(CRHR1). We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis.

Relationship between expression of IGFBP7 and clinicopathological variables in gastric cancer

AimsInsulin-like growth factor binding protein 7 (IGFBP7) is reported to have tumour suppressor function through an IGF-dependent pathway in various malignant tumours. However, the expression of IGFBP7 in adenocarcinoma and...

Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress.

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface.

Activated expression of AtWRKY53 negatively regulates drought tolerance by mediating stomatal movement.

AtWRKY53 is an early factor in drought response and activated expression of AtWRKY53 regulates stomatal movement via reduction of H 2 O 2 content and promotion of starch metabolism in guard cells. Drought is one of the most serious environmental factors limiting the productivity of agricultural crops worldwide. However, the mechanisms underlying drought tolerance in plants remain unclear. AtWRKY53 belongs to the group III of WRKY transcription factors. In this study, we observed both the mRNA and protein products of this gene are rapidly induced under drought conditions. Phenotypic analysis showed AtWRKY53 overexpression lines were hypersensitive to drought stress compared with Col-0 plants. The results of stomatal movement assays and abscisic acid (ABA) content detection indicated that the impaired stomatal closure of 53OV lines was independent of ABA. Further analysis found that WRKY53 regulated stomatal movement via reducing the H2O2 content and promoting the starch metabolism in guard cells. The results of quantitative real-time reverse transcriptase PCR showed that the expression levels of CAT2, CAT3 and QQS were up-regulated in 53OV lines. Chromatin immunoprecipitation assays demonstrated that AtWRKY53 can directly bind to the QQS promoter sequences, thus led to increased starch metabolism. In summary, our results indicated that the activated expression of AtWRKY53 inhibited stomatal closure by reducing H2O2 content and facilitated stomatal opening by promoting starch degradation.

RNA interference tools for the western flower thrips, Frankliniella occidentalis

The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips–tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced significantly fewer offspring compared to those in the dsRNA-GFP control group. Our findings indicate that an RNAi-based strategy to study gene function in thrips is feasible, can result in quantifiable phenotypes, and provides a much-needed tool for investigating the molecular mechanisms of thrips–tospovirus interactions. To our knowledge, this represents the first report of RNAi for any member of the insect order Thysanoptera and demonstrates the potential for translational research in the area of thrips pest control.

MALAT2-activated long noncoding RNA indicates a biomarker of poor prognosis in gastric cancer.

The purpose of this study is to investigate the role of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 2 (MALAT2) in the prognosis of stage II/III gastric cancer (GC) patients. The expression of MALAT2 was evaluated in cancer tissues from 146 stage II/III GC patients undergoing radical resection and 60 paired normal samples using quantitative real-time reverse transcriptase PCR. Differences in the expression of MALAT2 between 23 GC and paired normal colonic mucosa samples were analysed with the Wilcoxon test. Relationships between the expression level of MALAT2, patient clinico-pathological parameters and disease-free survival and overall survival were analysed using the uni-variate Kaplan-Meier method and the multivariate COX regression model. The quantitative polymerase chain reaction results showed that MALAT2 was frequently over-expressed in cancer tissues and this over-expression was found to significantly correlate with lymph node metastasis and tumor stage. The ectopic expression of MALAT2 contributed to the migration of human GC SGC-7901 cells, whereas knockdown of MALAT2 inhibited the migration of the SGC-7901 cells in vitro. Further investigation into the underlying mechanisms responsible for the migratory effects revealed that MALAT2 induced the epithelial-mesenchymal transition (EMT) through an MEK/extracellular signal-regulated kinase-dependent mechanism as treatment with the MEK inhibitor, U0126, decreased migration and reversed the EMT in the MALAT2 over-expressed SGC-7901 cells. The expression of MALAT2 is upregulated in GC tissues, and a higher expression level of MALAT2 might serve as a negative prognostic marker in stage II/III GC patients which implies the potential application of MALAT2 in the therapeutic treatment of GC.Cancer Gene Therapy advance online publication, 27 February 2015; doi:10.1038/cgt.2015.6.

Evidence of the internalization of animal caliciviruses via the roots of growing strawberry plants and dissemination to the fruit.

Human norovirus (NoV) is the leading cause of foodborne disease in the United States, and epidemiological studies have shown that fresh produce is one of the major vehicles for the transmission of human NoV. However, the mechanisms of norovirus contamination and persistence in fresh produce are poorly understood. The objective of this study is to determine whether human NoV surrogates, murine norovirus (MNV-1) and Tulane virus (TV), can attach and become internalized and disseminated in strawberries grown in soil. The soil of growing strawberry plants was inoculated with MNV-1 and TV at a level of 10(8) PFU/plant. Leaves and berries were harvested over a 14-day period, and the viral titer was determined by plaque assay. Over the course of the study, 31.6% of the strawberries contained internalized MNV-1, with an average titer of 0.81 ± 0.33 log10 PFU/g. In comparison, 37.5% of strawberries were positive for infectious TV, with an average titer of 1.83 ± 0.22 log10 PFU/g. A higher percentage (78.7%) of strawberries were positive for TV RNA, with an average titer of 3.15 ± 0.51 log10 RNA copies/g as determined by real-time reverse transcriptase quantitative PCR (RT-qPCR). In contrast, no or little virus internalization and dissemination were detected when TV was inoculated into bell peppers grown in soil. Collectively, these data demonstrate (i) virally contaminated soils can lead to the internalization of virus via plant roots and subsequent dissemination to the leaf and fruit portions of growing strawberry plants and (ii) the magnitude of internalization is dependent on the type of virus and plant.

Complement Factor I Promotes Progression of Cutaneous Squamous Cell Carcinoma

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase-PCR, and western blotting revealed marked overexpression of complement factor I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell-specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal-regulated kinase (ERK) 1/2 activation. Knockdown of CFI expression potently inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

BackgroundQuantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA.ResultsThe reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance.ConclusionsBayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0402-2) contains supplementary material, which is available to authorized users.

MiR-323-5p acts as a tumor suppressor by targeting the insulin-like growth factor 1 receptor in human glioma cells.

MicroRNAs, small noncoding RNA molecules, can regulate mammalian cell growth, apoptosis and differentiation by controlling the expression of target genes. The aim of this study was to investigate the function of miR-323-5p in the glioma cell line, U251. After over-expression of miR-323- 5p using miR-323-5p mimics, cell growth, apoptosis and migration were tested by MTT, flow cytometry and cell wound healing assay, respectively. We also assessed the influence of miR-323-5p on the mRNA expression of IGF- 1R by quantitative real-time reverse transcriptase PCR (qRT-PCR), and on the protein levels by Western blot analysi. In addition, dual-luciferase reporter assays were performed to determine the target site of miR-323-5p to IGF-1R 3'UTR. Our findings showed that over-expression of miR-323-5p could promote apoptosis of U251 and inhibit the proliferation and migration of the glioma cells. This study demonstrated that increased expression of miR-323-5p might be related to glioma progression, which indicates a potential role of miR-323-5p for clinical therapy.

TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer.

Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.

Dynamic changes in α- and β-amylase activities and gene expression in bulbs of the Oriental hybrid lily ‘Siberia’ during dormancy release

SUMMARYThe commercial production of cut lilies depends on a synchronous release of dormancy in bulbs kept under cold storage. We investigated the effects of storage at 4°C, 0°C, or -1.5°C, on dormancy release in bulbs of the Oriental hybrid Lilium ‘Siberia’ used for cut lily production. Cold storage at 4°C for 8 weeks resulted in full elongation of the central bud in bulbs and 100% shoot emergence within 3 weeks of planting, indicating complete release of dormancy. Starch breakdown, increased soluble sugar concentrations, and the induction of α-amylase activity occurred with significant changes in bulbs in week-8 of cold storage. Two cDNAs, one for the a-amylase gene, LiAMY, and the other for the β-amylase gene, LiBAM, were isolated and identified from ‘Siberia’ lily bulbs. Real-time reverse transcriptase quantitative PCR (RT-qPCR) analysis showed that expression of both LiAMY and LiBAM increased significantly in week-8 of cold storage. These changes were correlated with release from dormancy in commercial flowering bulbs of ‘Siberia’ lily.

Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR.

Gli proteins are transcription factors that mediate the transcriptional effects of Hedgehog signaling in vertebrates. The activities of Gli2 and Gli3 are regulated primarily by posttranslational modifications, while Gli1 is mostly regulated at the transcriptional level. Detection of endogenous Gli proteins had been hampered by lack of good antibodies, but this problem has been mostly resolved in recent years. In this chapter we describe methods of detecting expression of endogenous Gli genes in whole-cell lysates and in subcellular fractions and also provide protocols for the measurement of Gli mRNA levels by quantitative real-time reverse transcriptase PCR (qPCR).

Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

Circulating miR-125b as a biomarker of Ewing's sarcoma in Chinese children.

Previous studies indicated that microRNA-125b (miR-125b) has an important role in the progression of Ewing's sarcoma (ES). The purpose of the current study was to examine expression changes of miR-125b in the serum of ES patients and evaluate if the expression level of miR-125b could serve as a new biomarker for ES. This study was performed on patients who underwent surgical resection at our hospital between 2005 and 2013 after an initial diagnosis of ES. We measured serum miR-125b levels in 63 patients with ES and 126 healthy control patients using a real-time quantitative reverse transcriptase-PCR (qRT-PCR) method. Expression levels of serum miR-125b were distinctly decreased in ES patients when compared with healthy controls (P < 0.001). ES cases that had a poor response to chemotherapy presented a significant down-regulation of miR-125b (P = 0.001). The ROC curve showed that the serum miR-125b could serve as a valuable biomarker for differentiating ES patients from healthy controls with an AUC of 0.879 (95%CI = 0.817-0.924; P < 0.001). At a cut-off value of 2.203 for miR-125b, the sensitivity was 72.8% and the specificity was 87.2% in discriminating ES from the controls. Our results indicate that serum miR- 125b may serve as a useful noninvasive biomarker for ES.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis.EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control.MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects.In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. DisclosuresNo relevant conflicts of interest to declare.