Regulation Of Cyclooxygenase-2 Expression Research Articles

Targeting MEK/COX-2 axis improve immunotherapy efficacy in dMMR colorectal cancer with PIK3CA overexpression.

PIK3CA mutation or overexpression is associated with immunotherapy resistance in multiple cancer types, but is also paradoxically associated with benefit of COX-2 inhibition on patient survival of colorectal cancer (CRC) with mismatch repair deficiency (dMMR). This study examined whether and how PIK3CA status affected COX-2-mediated tumor inflammation and immunotherapy response of dMMR CRC. Murine colon cancer cells MC38, CT26, and CT26-Mlh1-KO were used to construct PIK3CA knockdown and overexpression models to mimic dMMR CRC with PIK3CA dysregulation, and xenograft models were used to evaluate how PIK3CA regulate COX-2 expression, CD8+ T cells infiltration, tumor growth, and therapy response to anti-PD-L1 treatment using immunocompetent mice. Western blot was carried out to delineate the signaling pathways in human and mouse cancer cells, and immunohistochemical analysis together with bioinformatics analysis using human patient samples. PIK3CA upregulates COX-2 expression through MEK/ERK signaling pathway independent of AKT signaling to promote tumor inflammation and immunosuppression. PIK3CA knockdown profoundly reduced CT26 tumor growth in a CD8+ T cell-dependent manner, while PIK3CA overexpression significantly inhibited CD8+ T cells infiltration and promoted tumor growth. Furthermore, MEK or COX-2 inhibition augmented the anti-tumor activity of anti-PD-L1 immunotherapy on dMMR CRC mouse models, accompanied with increased CD8+ T cells infiltration and activated tumor microenvironment. Our results identified that the PIK3CA hyperactivation in dMMR CRC upregulated COX-2 through MEK signaling, which inhibited CD8+ T cells infiltration and promoted tumor growth, together led to immunotherapy resistance. COX-2 or MEK inhibition may relieve therapy resistance and promote therapy efficacy of anti-PD-1/PD-L1 immunotherapy for treating dMMR CRC with PIK3CA overexpression or activating mutation.

Mycobacterium tuberculosis-Induced Prostaglandin J2 and 15-Deoxy-Prostaglandin J2 Inhibit Inflammatory Signals in Human M1 Macrophages via a Negative Feedback Loop.

Tuberculosis caused by Mycobacterium tuberculosis is a leading cause of death globally and a major health concern. In humans, macrophages are the first line invaded by M. tuberculosis. Upon infection, macrophages upregulate cyclooxygenase-2 (COX-2) expression and consequently elevate the formation of PGs, including PGE2 and PGD2. Although the role of proinflammatory PGE2 in M. tuberculosis infection has been reported, the roles of PGJ2 and 15-deoxy-PGJ2 (collectively named J2-PGs), the metabolites of PGD2 with anti-inflammatory features, remain elusive. In this study, we show that M. tuberculosis (H37Rv strain)-conditioned medium stimulates human monocyte-derived macrophages (MDMs) to elevate COX-2 expression along with robust generation of PGJ2, exceeding PGD2 formation, and to a minor extent also of 15-deoxy-PGJ2. Of interest, in M1-MDM phenotypes, PGJ2 and 15-deoxy-PGJ2 decreased M. tuberculosis (H37Rv strain)-conditioned medium-induced COX-2 expression and related PG formation by a negative feedback loop. Moreover, these J2-PGs downregulated the expression of the proinflammatory cytokines IL-6, IL-1β, and IFN-γ, but elevated the anti-inflammatory cytokine IL-10 and the M2 markers arginase-1 and CD163. These anti-inflammatory effects of J2-PGs in M1-MDM correlated with impaired activation of TGF-β-activated kinase 1/NF-κB/MAPK pathways. Finally, we found that J2-PGs regulate COX-2 expression, at least partially, via PGD2 receptor (DP1) and chemoattractant receptor homologue expressed on Th2 cells/DP2 receptors, but independent of the J2-PG receptor peroxisome proliferator-activated receptor-γ. Together, our findings reveal that M. tuberculosis induces COX-2 expression in human M1-MDMs, along with robust formation of J2-PGs that mediates anti-inflammatory effects via a negative feedback loop.

Circ_DLEU2 knockdown represses cell proliferation, migration and invasion, and induces cell apoptosis through the miR-582-5p/COX2 pathway in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant hematological neoplasm in adults. Researche indicates that circular RNAs (circRNAs) play paramount roles in the pathological process of AML. In this study, the role of circ_DLEU2 (circ_0000488) in AML is revealed. The expression of circ_DLEU2, microRNA-582-5p (miR-582-5p) and cyclooxygenase 2 (COX2) was determined by quantitative real-time PCR. Protein expression was detected by western blot. Cell proliferation was investigated by cell cycle, 5-Ethynyl-29-deoxyuridine and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays. Cell apoptosis was elucidated by apoptosis analysis assay. The targeting relationship between miR-582-5p and circ_DLEU2 or COX2 was predicted by the starbase online database, and identified by a dual-luciferase reporter assay. Circ_DLEU2 and COX2 expression were substantially up-regulated, while miR-582-5p was down-regulated in AML marrow samples and cells compared with control groups. Circ_DLEU2 knockdown suppressed cell proliferation, whereas it induced cell arrest at G0/G1 phase and cell apoptosis in AML; however, these effects were attenuated by miR-582-5p inhibitor. Additionally, circ_DLEU2 was associated with miR-582-5p, and miR-582-5p bound to COX2 in AML cells. Also, we found that circ_DLEU2 regulated COX2 expression by interacting with miR-582-5p. Circ_DLEU2 silencing hindered AML malignant progression via downregulating COX2 through sponging miR-582-5p. Our finding provides a theoretical basis for studying circRNA-directed therapy of AML.

Cyclooxygenases and platelet functions.

Cyclooxygenase (COX) isozymes, i.e., COX-1 and COX-2, are encoded by separate genes and are involved in the generation of the same products, prostaglandin (PG)G2 and PGH2 from arachidonic acid (AA) by the COX and peroxidase activities of the enzymes, respectively. PGH2 is then transformed into prostanoids in a tissue-dependent fashion due to the different expression of downstream synthases. Platelets present almost exclusively COX-1, which generates large amounts of thromboxane (TX)A2, a proaggregatory and vasoconstrictor mediator. This prostanoid plays a central role in atherothrombosis, as shown by the benefit of the antiplatelet agent low-dose aspirin, a preferential inhibitor of platelet COX-1. Recent findings have shown the relevant role played by platelets and TXA2 in developing chronic inflammation associated with several diseases, including tissue fibrosis and cancer. COX-2 is induced in response to inflammatory and mitogenic stimuli to generate PGE2 and PGI2 (prostacyclin), in inflammatory cells. However, PGI2 is constitutively expressed in vascular cells in vivo and plays a crucial role in protecting the cardiovascular systems due to its antiplatelet and vasodilator effects. Here, platelets' role in regulating COX-2 expression in cells of the inflammatory microenvironment is described. Thus, the selective inhibition of platelet COX-1-dependent TXA2 by low-dose aspirin prevents COX-2 induction in stromal cells leading to antifibrotic and antitumor effects. The biosynthesis and functions of other prostanoids, such as PGD2, and isoprostanes, are reported. In addition to aspirin, which inhibits platelet COX-1 activity, possible strategies to affect platelet functions by influencing platelet prostanoid receptors or synthases are discussed.

Regulation of Cyclooxygenase-2 Expression in Human T Cells by Glucocorticoid Receptor-Mediated Transrepression of Nuclear Factor of Activated T Cells.

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

PACER lncRNA regulates COX-2 expression in lung cancer cells.

Long noncoding RNAs (lncRNAs) are known to regulate gene expression; however, in many cases, the mechanism of this regulation is unknown. One novel lncRNA relevant to inflammation and arachidonic acid (AA) metabolism is the p50-associated COX-2 extragenic RNA (PACER). We focused our research on the regulation of PACER in lung cancer. While the function of PACER is not entirely understood, PACER is known to play a role in inflammation-associated conditions. Our data suggest that PACER is critically involved in COX-2 transcription and dysregulation in lung cancer cells.Our analysis of The Cancer Genome Atlas (TCGA) expression data revealed that PACER expression is significantly higher in lung adenocarcinomas than normal lung tissues. Additionally, we discovered that elevated PACER expression strongly correlates with COX-2 expression in lung adenocarcinoma patients. Specific siRNA-mediated knockdown of PACER decreases COX-2 expression indicating a direct relationship. Additionally, we show that PACER expression is induced upon treatment with proinflammatory cytokines to mimic inflammation. Treatment with prostaglandin E2 (PGE2) induces both PACER and COX-2 expression, suggesting a PGE2-mediated feedback loop. Inhibition of COX-2 with celecoxib decreased PACER expression, confirming this self-regulatory process. Significant overlap between the COX-2 promotor and the PACER promotor led us to investigate their transcriptional regulatory mechanisms. Treatment with pharmacologic inhibitors of NF-κB or AP-1 showed a modest effect on both PACER and COX-2 expression but did not eliminate expression. These data suggest that the regulation of expression of both PACER and COX-2 is complex and intricately linked.

Dihydrotestosterone regulation of cyclooxygenase-2 expression in bovine endometrial epithelium cells by androgen receptor mediated EGFR/PI3K/Akt pathway

Uterine prostaglandins F2α (PGF2α) is essential for implantation, initiation of luteolysis and delivery. Previous studies have demonstrated that the expression of Cyclooxygenase-2 (COX-2), an enzyme limiting PGF2α rate, is regulated by steroid hormones, and also dihydrotestosterone (DHT) may be involved in regulating COX-2 expression both positively and negatively. However, it remains unclear how whether DHT regulates COX-2 expression and consequent PGF2α release in bovine endometrial epithelial cells (EECs). In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in bovine endometria by immunohistochemistry, and investigated 5α-red1, 5α-red2, AR, and DHT levels at the different stages of endometria (follicle, early-, mid-, and late-pregnancy phases). The results showed that 5α-red1, 5α-red2 and AR all were expressed in endometria, and their expressions and the level of DHT significantly increased in the late-pregnancy phase compared with the mid-pregnancy phase. Moreover, we cultured EECs from the mid-pregnancy phase and the in vitro study showed that DHT dose-dependently increased COX-2 expression and PGF2a release, but AR antagonist (flutamide) inhibited the stimulating effect via DHT. In addition, the DHT-induced COX-2 expression and PGF2α release were subjected to the regulation of both EGFR/PI3K/Akt/NFkB signaling as the inhibitors of EGFR (AG1478) and PI3K/Akt (LY294002) and NFkB (QNZ) attenuated the DHT mediated effect. Taken together, the results demonstrated that DHT-induced COX-2 expression and consequent PGF2α release in bovine EECs were mediated through AR-derived EGFR transactivation and PI3K/Akt cascade leading to NFkB activation.

Ghrelin Regulates Cyclooxygenase-2 Expression and Promotes Gastric Cancer Cell Progression.

Aim To research the molecular mechanism of ghrelin in apoptosis, migratory, and invasion of gastric cancer (GC) cells. Methods After GC AGS cells were handled with ghrelin (10–8 M), cyclooxygenase-2 inhibitor NS398 (100 μM), and Akt inhibitor perifosine (10uM), the rates of apoptosis were detected by TUNEL assay and flow cytometry assay. We assessed the expressions of PI3K, p-Akt, and COX-2 proteins by making use of Western blot analysis. The cell migratory and invasion were detected by using wound-healing and transwell analysis. Results The migratory and invasion were increased in ghrelin-treated cells, while the rates of apoptosis were decreased. GC AGS cells treated with ghrelin showed an increase in protein expression of p-Akt, PI3K, and COX-2. After cells were treated with Akt inhibitor perifosine, the protein expression of p-Akt, PI3K, and COX-2 and the cell migratory, invasion, and apoptosis were partly recovered. After cells were treated with cyclooxygenase-2 inhibitor NS398, the protein expression of COX-2 and the cell migratory and invasion were decreased, while the rates of apoptosis were increased. Conclusion Ghrelin regulates cell migration, invasion, and apoptosis in GC cells through targeting PI3K/Akt/COX-2. Ghrelin increases the expression of COX-2 in GC cells by targeting PI3K/Akt. Ghrelin is suggested to be one of the molecular targets in GC.

LncRNA-PACER upregulates COX-2 and PGE2 through the NF-κB pathway to promote the proliferation and invasion of colorectal-cancer cells.

Backgroundp50-associated cyclooxygenase-2 extragenic RNA (PACER) is a recently identified antisense long non-coding RNA (lncRNA) located on the upstream of the promoter region of cyclooxygenase-2 (COX-2). Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells. However, the role of this lncRNA in colorectal cancer (CRC) remains elusive. Here, we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.MethodsReal-time quantitative PCR (RT-qPCR) analysis was used to evaluate the expression of PACER in CRC tissues and cells. Methyl thiazolyl tetrazolium (MTT) analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation. The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays, colony-formation assay, and transwell assays. We then used fluorescence in situ hybridization (FISH) to establish the subcellular localization of PACER. COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER. Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER. Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo. Enzyme-linked immunosorbent assay (ELISA) was then used to quantify prostaglandin E2 (PGE2) production upon knock-down of PACER.ResultsRT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells, and a high PACER-expression level was associated with poor prognosis. MTT assay, wound-healing assay, colony-formation assay, and transwell assay revealed that PACER enhanced CRC-cell proliferation, invasion, and metastasis in vitro. Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus. RT-qPCR showed that PACER increased mRNA levels of COX-2. Western blot analysis demonstrated, under normal circumstances, that knock-down of PACER decreased the COX-2 protein level. In the case of p50 absence, COX-2 protein increased rapidly and remained highly expressed after knocking down PACER. Luciferase assay revealed that PACER modulated the COX-2 promoter region. Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo. ELISA revealed that PACER knock-down inhibited PGE2 production.ConclusionsPACER modulates COX-2 expression through the nuclear factor kappa B (NF-κB) pathway in CRC. An increased level of PACER enhances proliferation, migration, and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.

Chitosan nanoparticles loaded with aspirin and 5-fluororacil enable synergistic antitumour activity through the modulation of NF-κB/COX-2 signalling pathway.

Based on the enhancement of synergistic antitumour activity to treat cancer and the correlation between inflammation and carcinogenesis, the authors designed chitosan nanoparticles for co-delivery of 5-fluororacil (5-Fu: an as anti-cancer drug) and aspirin (a non-steroidal anti-inflammatory drug) and induced synergistic antitumour activity through the modulation of the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2) signalling pathways. The results showed that aspirin at non-cytotoxic concentrations synergistically sensitised hepatocellular carcinoma cells to 5-Fu in vitro. It demonstrated that aspirin inhibited NF-κB activation and suppressed NF-κB regulated COX-2 expression and prostaglandin E2 (PGE2) synthesis. Furthermore, the proposed results clearly indicated that the combination of 5-Fu and aspirin by chitosan nanoparticles enhanced the intracellular concentration of drugs and exerted synergistic growth inhibition and apoptosis induction on hepatocellular carcinoma cells by suppressing NF-κB activation and inhibition of expression of COX-2.

NF-κB "decoy" inhibits COX-2 expression in epileptic rat brain.

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.

Selective inhibition of cyclooxygenase-2 protects porcine aortic endothelial cells from human antibody-mediated complement-dependent cytotoxicity.

Cyclooxygenase-2 (COX-2) is an inducible enzyme with catalytic activity for biosynthesis of prostaglandins which are the key mediators of inflammation. COX-2 is also the therapeutic target for widely used non-steroidal anti-inflammatory drugs (NSAIDs). However, the involvement of COX-2 in xenotransplantation (eg, pig-to-non-human primate) remains poorly recognized. We investigatedthe mechanisms that regulate COX-2 expression and the effects of COX-2 on porcine aortic endothelial cell (PAEC) viability using in vitro pig-to-primate xenotransplantation modelandin vivopig-to-mouse cellular transplant model. Regulation of COX-2 expression was assessed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. The effects of inhibition or downregulation of COX-2 on PAEC viability were assessed by propidium iodide (PI)-Annexin V staining and Cell Counting Kit-8 assay. Human serum triggered robust COX-2 expression in PAECs in a dose- and time-dependent manner. Induction of COX-2 expression by human serum was partially through activation of both canonical and non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) signaling and increasing intracellular calcium. Cytokines like tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), IL-17, were able to induce COX-2 expression. Selective inhibition of COX-2 by celecoxib dramatically decreased PAEC death in vitro and in vivo as defined by propidium iodide (PI)-Annexin V staining. Consistently, downregulation of COX-2 expression by NF-κb inhibitors or calcium chelator BAPTA decreased human serum-induced PAEC death as well. Silencing of COX-2 expression by small interfering RNA (siRNA) protected PAEC viability when transplanted under kidney capsule of C57BL/6 mice. Taken together, our data suggest that COX-2 is highly induced in PAECs by xenogenic serum and associated with human antibody-mediated complement-dependent cytotoxicity. COX-2 might be a potential therapeutic target to improve xenotransplantation.

Significance of Cyclooxygenase-2 in Oncogenesis.

Abstract The cyclooxygenase-2 (COX-2) enzyme catalyses the first stage of biosynthesis of prostanoids, proteins that are implicated in various physiological and pathological processes in humans and animals. The expression of COX-2 increases significantly during pathological processes accompanied by inflammation, pain and fever. Overexpression of COX-2 was determined in tumour tissues, which suggests that this enzyme participates in oncogenesis. In this paper the topics discussed are mechanisms regulating COX-2 expression, COX isoforms, their role in the body and the oncogenic mechanisms triggered by the overexpression of COX-2, including inhibition of apoptosis, intensification of neoangiogenesis, increased metastatic capacity, and weakening of the immune system. The significance of and the mechanisms by which COX-2 participates in oncogenesis have been studied intensively in recent years. The results are highly promising, and they expand our understanding of the complex processes and changes at the molecular, cellular and tissue level that promote oncogenesis and cancer progression. Notwithstanding the knowledge already gleaned, many processes and mechanisms have not yet been elucidated in human medicine and, in particular, in veterinary medicine. Further research is required to develop effective tumour diagnostic methods and treatment procedures for humans and animals.

Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Human amnion fibroblasts produce abundant prostaglandin E2 (PGE2), which plays a crucial role in parturition by stimulating not only myometrial contraction and cervical ripening but also the expression of the rate-limiting enzyme in PGE2 synthesis-namely, cyclooxygenase-2 (COX-2). This feed-forward induction of COX-2 expression by PGE2 is mediated via its receptors coupled with the cAMP and PKA pathway and subsequent phosphorylation of the transcription factors cAMP-response element binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3). Although prostaglandin E receptor (EP)-2 and EP4 for PGE2 are coupled with activation of the cAMP and PKA pathway, the exact roles of these 2 receptors in the regulation of COX-2 expression in amnion fibroblasts remain to be determined. Here, we clarify this issue by employing human amnion tissue and fibroblasts with the long-term objective of specific targeting of prostaglandin synthesis in prevention of preterm birth. We find that an EP2 agonist caused long-lasting increases in CREB phosphorylation and COX-2 expression, whereas an EP4 agonist induced only transient increases in CREB phosphorylation and COX-2 expression in amnion fibroblasts. Moreover, only EP2 stimulation increased STAT3 phosphorylation, whereas only EP4 stimulation increased PI3K activity. EP4 antagonist or inhibition of PI3K enhanced the induction of CREB and STAT3 phosphorylation and COX-2 expression by PGE2 or EP2 stimulation, which was attenuated by EP4 overexpression. Of interest, PGE2 and cortisol, both well-demonstrated stimulants of COX-2 expression in amnion fibroblasts, increased EP2 but decreased EP4 receptor expression. Furthermore, increased EP2 but decreased EP4 abundance were observed in amnion tissue at parturition. We conclude that EP2 and EP4 receptors play different roles in the regulation of COX-2 expression in human amnion fibroblasts. EP2 is the dominant PGE2 receptor mediating the induction of COX-2 at parturition, which can be attenuated by simultaneous activation of PI3K coupled to the EP4 receptor.-Lu, J.-W., Wang, W.-S., Zhou, Q., Gan, X.-W., Myatt, L., Sun, K. Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Inhibitory effect of bromfenac sodium hydrate ophthalmic solution on alkali burn induced-corneal neovascularization in rats

Objective To investigate the inhibitory effect of bromfenac sodium hydrate ophthalmic solution on corneal neovascularization (CNV) induced by alkali burn. Methods A total of 192 specific pathogen free (SPF) degree adult male Sprague-Dawley (SD) rats were used in this study.One hundred and seventy-two rats were chosen to establish CNV model with alkali burn in the right eyes.Following alkali burn, rats were randomly divided into CNV group, model control group, bromfenac sodium group and fluorometholone group, with 43 rats (43 eyes) in each group.Another 20 rats (40 eyes) served as normal control group.One day after modeling, the model control group, bromfenac sodium group and fluorometholone group received phosphate buffer saline (PBS), bromfenac sodium hydrate ophthalmic solution and 0.1% fluorometholone eye drops, respectively.The state of cornea and anterior chamber and the growth of CNV of rats in each group were observed by slit-lamp microscope every day after modeling.At 1, 3, 7, 14, 21 and 28 days after modeling, the anterior segment photos of the experimental eyes were captured, and the percent of cornea areas covered by CNV was calculated.At 7, 14 and 28 days after modeling, the eye tissue sections were stained with hematoxylin and eosin staining and immunohistochemistry staining to evaluate the expressions of CD45 and VEGF-A.Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of COX-2 and VEGF mRNA and protein level.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO). Results Each model group showed corneal edema and opacification 1 day after modeling.The corneal edema was aggravated 7 days after modeling.On the 14th day after modeling, the degree of corneal opacity and edema decreased gradually.On the 28th day after modeling, leucoma was observed in CNV group and model control group, and nebula was observed in bromfenac sodium group and fluorometholone group.At 7, 14, 21 and 28 days after modeling, the percentages of CNV areas in bromfenac sodium group and fluorometholone group were significantly lower than those in CNV group and model control group (all at P 0.05). On the 7th day after modeling, the thinning of corneal epithelial layer, edema and arrangement disorder of stroma layer were observed, and the expression of VEGF-A was positive in all model groups; a small amount of CD45 positive inflammatory cell infiltrations were observed in CNV group and model control group.On the 14th and 28th day after modeling, CNV was seen in the center of cornea in CNV group and model control group; the epithelial keratosis and reduction of corneal edema were seen in each group, and no inflammatory cell infiltration was observed in each group.On the 7th day after modeling, the expressions of COX-2 and VEGF mRNA in CNV group and model control group were significantly higher than those in normal control group, bromfenac sodium group and fluorometholone group (all at P<0.05), the expressions of COX-2 and VEGF protein in bromfenac sodium group were significantly lower than those in CNV group (all at P<0.05). The corneal perforation rate in model control group and bromfenac sodium group was 10% (1 case in 10 rats). The corneal perforation rate in fluorometholone group was 30% (3 cases in 10 rats). In each model group, 10% to 30% rats had hyphema. Conclusions Bromfenac sodium hydrate ophthalmic solution can inhibit the formation and growth of CNV after alkali burn in rats.This effect may be mediated by regulating COX-2 expression, reducing inflammation and inhibiting VEGF production. Key words: Corneal neovascularization; Alkali burn; Bromfenac sodium; Cyclooxygenase-2

PVT1 (rs13281615) and miR-146a (rs2910164) polymorphisms affect the prognosis of colon cancer by regulating COX2 expression and cell apoptosis.

In this study, we aimed to investigate the potential correlation between rs13281615/rs2910164 polymorphisms and the prognosis of colon cancer (CC). Taqman was utilized to genotype the rs13281615/rs2910164 polymorphisms in recruited subjects. Kaplan-Meier survival curves were calculated to study the prognostic values of different genotypes of rs13281615/rs2910164 polymorphisms. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays were conducted to establish a potential signaling pathway underlying the role of rs13281615/rs2910164 polymorphisms, whereas bioinformatics analysis and luciferase reporter assays were performed to identify plasmacytoma variant translocation 1 (PVT1) and cyclooxygenase-2 (COX2) as targets of microRNA-146a (miR-146a). No significant difference was observed in respect to clinical characteristics among subjects with different genotypes. However, patients genotyped as GG/CC + GC showed the lowest chance of survival, whereas patients of GA + AA/GG genotype showed the highest chance of survival. Moreover, the relative expressions of PVT1, prostaglandin E2 (PGE2), and COX2 were the lowest and the relative expression of miR-146a was the highest in GA + AA/GG subjects, validating the roles of PVT1, miR-146a, and COX2 in CC. In addition, both PVT1 and COX2 were identified as virtual targets of miR-146a, and the luciferase activities of cells cotransfected with wild-type PVT1/COX2 and miR-146a mimics were significantly reduced. Moreover, the presence of PVT1 decreased the level of miR-146a whereas increasing the messenger RNA and protein levels of COX2, thus establishing a PVT1/miR-146a/COX2 signaling pathway underlying the pathogenesis of CC. The presence of rs13281615 G > A polymorphism on PVT1 and the rs2910164 C > G polymorphism on miR-146a contributes to a favorable prognosis in CC patients via modulating the activity of the PVT1/miR-146a/COX2 signaling pathway.

MiR-155 Modulates Cockroach Allergen- and Oxidative Stress-Induced Cyclooxygenase-2 in Asthma.

Exposure to cockroach allergen is a strong risk factor for developing asthma. Asthma has been associated with allergen-induced airway epithelial damage and heightened oxidant stress. In this study, we investigated cockroach allergen-induced oxidative stress in airway epithelium and its underlying mechanisms. We found that cockroach extract (CRE) could induce reactive oxygen species (ROS) production, particularly mitochondrial-derived ROS, in human bronchial epithelial cells. We then used the RT2 Profiler PCR array and identified that cyclooxygenase-2 (COX-2) was the most significantly upregulated gene related to CRE-induced oxidative stress. miR-155, predicted to target COX-2, was increased in CRE-treated human bronchial epithelial cells, and was showed to regulate COX-2 expression. Moreover, miR-155 can bind COX-2, induce COX-2 reporter activity, and maintain mRNA stability. Furthermore, CRE-treated miR-155-/- mice showed reduced levels of ROS and COX-2 expression in lung tissues and PGE2 in bronchoalveolar lavage fluid compared with wild-type mice. These miR-155-/- mice also showed reduced lung inflammation and Th2/Th17 cytokines. In contrast, when miR-155-/- mice were transfected with adeno-associated virus carrying miR-155, the phenotypic changes in CRE-treated miR-155-/- mice were remarkably reversed, including ROS, COX-2 expression, lung inflammation, and Th2/Th17 cytokines. Importantly, plasma miR-155 levels were elevated in severe asthmatics when compared with nonasthmatics or mild-to-moderate asthmatics. These increased plasma miR-155 levels were also observed in asthmatics with cockroach allergy compared with those without cockroach allergy. Collectively, these findings suggest that COX-2 is a major gene related to cockroach allergen-induced oxidative stress and highlight a novel role of miR-155 in regulating the ROS-COX-2 axis in asthma.

Abstract 4438: NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells

Abstract Sodium butyrate (NaBt) is a histone deacetylase inhibitor (HDACi) produced in the colon by commensal microbiota-mediated fermentation of dietary fibers. It is regarded to have tumor suppressive and anti-inflammatory effects. NaBt can regulate gene expression through chromatin remodeling and altered transcription. However, it has not been addressed adequately whether inflammatory genes may also be regulated by NaBt in a post transcriptional manner via AU rich elements (ARE) in their 3'UTR. Overexpression of Cyclooxygenase-2 (COX-2) is observed in chronic inflammatory diseases and colorectal cancer (CRC). The aim of this study was to explore whether NaBt regulates COX-2 expression via post-transcriptional mechanisms. Pre-confluent Caco-2 and HT-29 cells were treated with different concentrations (1-5mM) of NaBt for short (3-6h) and long (24-48h) time points. Gene expression was determined by qPCR or Western blot. mRNA stability was determined with an Actinomycin D (ActD) chase assay. 3'UTR activity was determined in an RPSM30 vector containing the ARE rich and corresponding mutated regions of the 3'UTR from COX-2. NaBt strongly reduced COX-2 mRNA and protein expression with both short and long-term treatments. This reduction was not through NF-κB, since short-term treatment of cells with NaBt could not alter NF-κB activity in a luciferase reporter assay. An Act D chase assay indicated that in the absence of new mRNA synthesis, NaBt treatment induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. However, we did not observe any change in the 3'UTR activity of COX-2 in the presence of NaBt alone, indicating that the presence of a stress factor (such as ActD) was necessary for mRNA stabilization. Treatment of Caco-2 cells with NaBt and ActD together resulted in enhanced activation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of the ARE binding protein HuR. This was also reflected in increased COX-2 mRNA and protein levels. In HT-29 cells, simultaneous (NaBt+ActD) treatment resulted in a decrease in p-MK2, but an increase in p-Chk2 compared to ActD alone. p-Chk2 can preserve Cdk1 in a hyperphosphorylated state where it may promote retention of HuR in the nucleus, facilitating COX2 mRNA degradation and lower protein levels. No change in p-Chk2 was observed in Caco-2 cells in a similar experimental set up. These findings suggest that in the presence of a stress-inducing agent like ActD, NaBt can alter mRNA stability of COX-2 in a contextual manner, dependent on upstream signaling. Thus, while the rate of COX-2 mRNA degradation in HT-29 was enhanced, it slows down the same in Caco-2 cells. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of stress. Citation Format: Sreeparna Banerjee, Shabnam Enayat, Sinem Tuncer, Doğukan H. Ulgen, Aydan Torun. NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4438.

Epigenetic regulation of COX-2 expression by DNA methylation via NF-KB activation in ketamine-induced ulcerative cystitis

Epigenetic regulation of COX-2 expression by DNA methylation via NF-KB activation in ketamine-induced ulcerative cystitis

XRCC5 cooperates with p300 to promote cyclooxygenase-2 expression and tumor growth in colon cancers.

Cyclooxygenase (COX) is the rate-limiting enzyme in prostaglandins (PGs) biosynthesis. Previous studies indicate that COX-2, one of the isoforms of COX, is highly expressed in colon cancers and plays a key role in colon cancer carcinogenesis. Thus, searching for novel transcription factors regulating COX-2 expression will facilitate drug development for colon cancer. In this study, we identified XRCC5 as a binding protein of the COX-2 gene promoter in colon cancer cells with streptavidin-agarose pulldown assay and mass spectrometry analysis, and found that XRCC5 promoted colon cancer growth through modulation of COX-2 signaling. Knockdown of XRCC5 by siRNAs inhibited the growth of colon cancer cells in vitro and of tumor xenografts in a mouse model in vivo by suppressing COX-2 promoter activity and COX-2 protein expression. Conversely, overexpression of XRCC5 promoted the growth of colon cancer cells by activating COX-2 promoter and increasing COX-2 protein expression. Moreover, the role of p300 (a transcription co-activator) in acetylating XRCC5 to co-regulate COX-2 expression was also evaluated. Immunofluorescence assay and confocal microscopy showed that XRCC5 and p300 proteins were co-located in the nucleus of colon cancer cells. Co-immunoprecipitation assay also proved the interaction between XRCC5 and p300 in nuclear proteins of colon cancer cells. Cell viability assay indicated that the overexpression of wild-type p300, but not its histone acetyltransferase (HAT) domain deletion mutant, increased XRCC5 acetylation, thereby up-regulated COX-2 expression and promoted the growth of colon cancer cells. In contrast, suppression of p300 by a p300 HAT-specific inhibitor (C646) inhibited colon cancer cell growth by suppressing COX-2 expression. Taken together, our results demonstrated that XRCC5 promoted colon cancer growth by cooperating with p300 to regulate COX-2 expression, and suggested that the XRCC5/p300/COX-2 signaling pathway was a potential target in the treatment of colon cancers.