253 GENE SILENCING OF CYCLOOXYGENASE-2 mRNA BY RNA INTERFERENCE IN BOVINE CUMULUS - GRANULOSA CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 234
Author(s):  
S.-I. Kobayashi ◽  
M. Sakatani ◽  
S. Kobayashi ◽  
K. Okuda ◽  
M. Takahashi
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Mrna Expression ◽  
Culture Medium ◽  
Cyclooxygenase 2 ◽  
Target Cells ◽  
Specific Gene ◽  
Cox 2 ◽  
Sirna Group ◽  
Interference Rna

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F2� (PGF2�) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF2� concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF2� concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF2� concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells. This study was supported by the Japan Society for the Promotion of Science for Young Scientists.

scholarly journals Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 454
Author(s):  
Jaime Palomino ◽  
Javiera Flores ◽  
Georges Ramirez ◽  
Victor H. Parraguez ◽  
Monica De los Reyes
Keyword(s):  
Gene Expression ◽  
Bone Morphogenetic Protein ◽  
Cyclooxygenase 2 ◽  
Oestrous Cycle ◽  
Progesterone Concentration ◽  
Plasma Progesterone ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Bone Morphogenetic Protein 15 ◽  
Cox 2

The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.

scholarly journals Cyclooxygenase-2 (COX-2) mRNA Expression Levels in Normal Lung Tissues and Non-small Cell Lung Cancers

1999 ◽  
Vol 90 (12) ◽  
pp. 1338-1373 ◽  
Author(s):  
Mari Ochiai ◽  
Tetsuya Oguri ◽  
Takeshi Isobe ◽  
Shinichi Ishioka ◽  
Michio Yamakido
Keyword(s):  
Mrna Expression ◽  
Small Cell ◽  
Cyclooxygenase 2 ◽  
Normal Lung ◽  
Small Cell Lung ◽  
Lung Cancers ◽  
Expression Levels ◽  
Cox 2 ◽  
Mrna Expression Levels

Regulation of macrophage cyclooxygenase-2 gene expression by modifications of histone H3

2004 ◽  
Vol 286 (5) ◽  
pp. L956-L962 ◽  
Author(s):  
Gye Young Park ◽  
Myungsoo Joo ◽  
Tetyana Pedchenko ◽  
Timothy S. Blackwell ◽  
John W. Christman
Keyword(s):  
Gene Expression ◽  
Chromatin Structure ◽  
Gene Activation ◽  
Histone H3 ◽  
Major Effect ◽  
Cyclooxygenase 2 ◽  
Raw 264.7 Cells ◽  
Transcriptional Level ◽  
Sequence Motif ◽  
Cox 2

Some transcription factors involved in the regulation of cyclooxygenase 2 (COX-2) expression in macrophage, including NF-κB, interact with p300, which contains histone acetyltransferase (HAT) enzyme complex. Chromatin structure is regulated by modifying enzymes, including HAT, and plays an important role in eukaryotic gene regulation through histone modification. We hypothesized that changes in chromatin structure related to phosphorylation and acetylation of histone H3 adjacent to key DNA binding sequence motif in the COX-2 promoter contribute to COX-2 gene activation in macrophages. Sodium butyrate (NaBT) is a short-chain fatty acid that possesses histone deacetyltransferase-inhibiting activity. Our data show that NaBT accentuates LPS-induced COX-2 gene expression at a transcriptional level, even though NaBT alone does not induce the COX-2 gene expression. Using a chromatin immunoprecipitation assay, we showed that costimulation of RAW 264.7 cells with NaBT and LPS synergistically increases COX-2 gene expression through both acetylation and phosphorylation of histone H3 at the promoter site. Our data show that NaBT accentuates LPS-induced COX-2 gene expression through MAP kinase-dependent increase of phosphorylation and acetylation of histone H3 at the COX-2 promoter site. These data indicate that posttranslational modification of histone H3 has a major effect on COX-2 gene expression by macrophages.

scholarly journals ApoL1 renal risk variants induce aberrant THP-1 monocyte differentiation and increase eicosanoid production via enhanced expression of cyclooxygenase-2

2018 ◽  
Vol 315 (1) ◽  
pp. F140-F150 ◽  
Author(s):  
Hewang Lee ◽  
Hila Roshanravan ◽  
Ying Wang ◽  
Koji Okamoto ◽  
Junghwa Ryu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Inhibitor ◽  
Cyclooxygenase 2 ◽  
Prostaglandin E ◽  
Monocyte Differentiation ◽  
Eicosanoid Production ◽  
Gene And Protein Expression ◽  
Cox 2 ◽  
Tgf Β1

Apolipoprotein L1 ( ApoL1) genetic variants are strongly associated with kidney diseases. We investigated the role of ApoL1 variants in monocyte differentiation and eicosanoid production in macrophages, as activated tissue macrophages in kidney might contribute to kidney injury. In human monocyte THP-1 cells, transient overexpression of ApoL1 (G0, G1, G2) by transfection resulted in a 5- to 11-fold increase in CD14 and CD68 gene expression, similar to that seen with phorbol-12-myristate acetate treatment. All ApoL1 variants caused monocytes to differentiate into atypical M1 macrophages with marked increase in M1 markers CD80, TNF, IL1B, and IL6 and modest increase in the M2 marker CD163 compared with control cells. ApoL1-G1 transfection induced additional CD206 and TGFB1 expression, and ApoL1-G2 transfection induced additional CD204 and TGFB1 expression. Gene expression of prostaglandin E2 (PGE2) synthase and thromboxane synthase and both gene and protein expression of cyclooxygenase-2 (COX-2) were increased by ApoL1-G1 and -G2 variants compared with -G0 transfection. Higher levels of PGE2 and thromboxane B2, a stable metabolite of thromboxane A2, and transforming growth factor (TGF)-β1 were released into the supernatant of cultured THP-1 cells transfected with ApoL1-G1 and -G2, but not -G0. The increase in PGE2, thromboxane B2, and TGF-β1 was inhibited by COX-2-specific inhibitor CAY10404 but not by COX-1-specific inhibitor SC-560. These results demonstrate a novel role of ApoL1 variants in the regulation of monocyte differentiation and eicosanoid metabolism, which could modify the immune response and promote inflammatory signaling within the local targeted organs and tissues including the kidney.

scholarly journals Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
René F. Ketting
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Gene Silencing ◽  
Small Rnas ◽  
Self Regulation ◽  
Fine Tuning ◽  
Specific Gene ◽  
Next Generation ◽  
C Elegans ◽  
Argonaute Proteins

AbstractEndogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we find that sRNA abundance, the pattern of origin of sRNA and 3’ UTR length are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we discovered that ALG-3- and ALG-4-bound 26G-RNAs are dampening the expression of their own mRNAs, revealing a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.Author SummarySmall RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are important to initiate specific gene expression programs in the next generation. The nematode Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs that are divided into two subpopulations: one expressed in the spermatogenic gonad and another expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression.

Fragile X-related protein 1 (FXR1) regulates cyclooxygenase-2 (COX-2) expression at the maternal–fetal interface

2018 ◽  
Vol 30 (11) ◽  
pp. 1566 ◽  
Author(s):  
Xiao-Cui Li ◽  
Meng-fan Song ◽  
Feng Sun ◽  
Fu-Ju Tian ◽  
Yu-mei Wang ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Recurrent Miscarriage ◽  
Fragile X ◽  
Firefly Luciferase ◽  
Cyclooxygenase 2 ◽  
Luciferase Reporter ◽  
Related Protein ◽  
Expression Levels ◽  
Cox 2 ◽  
Mrna Expression Levels

Cyclooxygenase-2 (COX-2) is regulated post-transcriptionally by the AU-rich element (ARE) in the 3′-untranslated region (UTR) of its mRNA. However, the mechanism of COX-2 induction in infertility has not been thoroughly elucidated to date. The aim of this study was to examine the association between COX-2 and fragile X-related protein 1 (FXR1) in trophoblasts. Using quantitative reverse transcription polymerase chain reaction, our results showed that FXR1 mRNA expression levels were significantly decreased in trophoblasts from recurrent miscarriage patients compared with healthy controls; conversely, COX-2 mRNA expression levels were increased in patient samples. We also observed that FXR1 was highly expressed in human placental villi during early pregnancy. Furthermore, we used western blotting and immunofluorescence to analyse the expression levels of FXR1 and COX-2 in HTR-8 cells that were treated with tumour necrosis factor α; we observed that the expression of COX-2 was clearly increased in HTR-8 cells treated with FXR1 small interfering RNA, whereas the expression of COX-2 was effectively decreased in HTR-8 cells with FXR1 overexpressed via a plasmid. Importantly, bioinformatics analysis identified FXR1 binding sites in the 3′-UTR region of COX-2 and firefly luciferase reporter assay analysis verified that FXR1 binds directly to the 3′-UTR region of COX-2. ELISA assays showed that overexpression of FXR1 enhanced vascular endothelial growth factor-A and interleukin-8 expression in HTR-8 cells, whereas conversely, knockdown of FXR1 effectively repressed these effects. In conclusion, the results of this study indicate that FXR1 is a novel COX-2 regulatory factor.

Effect of Omega-3 Fatty Acids on Prostaglandin F2alpha-Induced Cyclooxygenase-2 (Cox-2) Gene Expression in Bovine Luteal Cells In Vitro.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 211-211
Author(s):  
Nicole R. White ◽  
Patrick D. Burns ◽  
Jirapat Charumilinda ◽  
Andrew D. Bryant ◽  
Zane T. Prosser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Cyclooxygenase 2 ◽  
Omega 3 Fatty Acids ◽  
Omega 3 ◽  
Prostaglandin F2alpha ◽  
Luteal Cells ◽  
Cox 2
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