P–307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A C Mestr. Citrinovitz ◽  
J Jauckus ◽  
J Hauke ◽  
C D Langhans ◽  
K Schwarz ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mrna Expression ◽  
Total Protein ◽  
Metabolic Pathways ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Endometrial Stromal Cells ◽  
Oxidation Pathway ◽  
Mrna Expression Levels

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S–239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings: The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number Not applicable

P-307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A C Mestre Citrinovitz ◽  
J Jauckus ◽  
J Hauke ◽  
C D Langhans ◽  
K Schwarz ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mrna Expression ◽  
Total Protein ◽  
Metabolic Pathways ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Endometrial Stromal Cells ◽  
Oxidation Pathway ◽  
Mrna Expression Levels

Abstract Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S-239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number not applicable

scholarly journals Therapeutic Potential of Thrombomodulin in Renal Fibrosis of Nephrotoxic Serum Nephritis in Wistar-Kyoto Rats

2020 ◽  
Vol 45 (3) ◽  
pp. 391-406
Author(s):  
Nobuhiro Kanazawa ◽  
Masayuki Iyoda ◽  
Shohei Tachibana ◽  
Kei Matsumoto ◽  
Yukihiro Wada ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Renal Fibrosis ◽  
Therapeutic Potential ◽  
The Body ◽  
Expression Levels ◽  
Nephrotoxic Serum Nephritis ◽  
Collagen Iii ◽  
Wistar Kyoto ◽  
Mrna Expression Levels

Background: Recombinant human soluble thrombomodulin (rhTM) was approved in 2008 and has been used for treatment of disseminated intravascular coagulation in Japan. The antifibrotic effects of rhTM in acute exacerbation of idiopathic pulmonary fibrosis are well established, but the therapeutic potential of rhTM in renal fibrosis remains poorly understood. Methods: Nephrotoxic serum nephritis (NTS-N) was induced in 22 female Wistar-Kyoto (WKY) rats on day 0. Rats were administered either rhTM or vehicle intraperitoneally, every day from day 4 to day 55. Rats were sacrificed on day 56 when renal fibrosis was established and renal morphological investigations were performed. In vitro, rat renal fibroblasts (NRK-49F) were pretreated with rhTM or saline, and expression levels of profibrogenic gene induced by thrombin were analyzed by real-time reverse transcription polymerase chain reaction. Results: Compared to WKY-GN-vehicle rats, the body weights of WKY-GN-rhTM rats were significantly greater on day 55. By day 56, rhTM had significantly reduced serum creatinine levels in NTS-N. On the other hand, urinary protein excretion was comparable between the two treatment groups throughout the study. The percentage of Masson trichrome-positive areas in WKY-GN-rhTM rats was significantly lower compared to that in WKY-GN-vehicle rats. Glomerular fibrin deposition was significantly reduced in WKY-GN-rhTM rats. In addition, rhTM significantly reduced the renal cortical mRNA expression levels of TNF-α, Toll-like receptor 4, MYD88, TGF-β, αSMA, collagen I, collagen III, fibronectin, and protease-activated receptor 1 (PAR1), a thrombin receptor. In vitro, thrombin stimulation of NRK-49F cells significantly enhanced the mRNA expression levels of αSMA and PAR1, and these upregulations were significantly reduced by pretreatment with rhTM. Conclusions: Administration of rhTM after establishment of crescentic glomerulonephritis (GN) attenuated the subsequent development of renal fibrosis in NTS-N, possibly in part by inhibiting thrombin-mediated fibrogenesis. Our results suggest that rhTM may offer a therapeutic option for limiting the progression of chronic kidney disease in crescentic GN.

Expression of Ara-C Metabolizing Enzymes Mediates in Vitro Sensitivity to Ara-C in Primary AML Cells From Patients with Denovo AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3481-3481
Author(s):  
Ajay Abraham ◽  
Savitha Varatharajan ◽  
Ashok kumar Jayavelu ◽  
Shaji R Velayudhan ◽  
Rayaz Ahmed ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Candidate Genes ◽  
P Value ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutation Status ◽  
Expression Levels ◽  
In Vitro Sensitivity ◽  
Mrna Expression Levels

Abstract Abstract 3481 Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of pro-drug Ara-C is the enzymatic conversion to its active tri-phosphorylated form ara-CTP. Many drug activating (Deoxycytidine kinase (dCK) and human Equilibrative Nucleoside Transporter 1 (hENT1) and deactivating (Cytidine deaminase (CDA), 5'nucleotidase (NT5C2) genes and ribonucleoside reductase (RRM1), which are involved in transport and biotransformation of cytarabine contribute to the variation in ara-C sensitivity in AML patients. FLT3-ITD and NPM1 mutations act as major poor and good prognostic markers respectively in cytogenetically normal AML. The effect of these mutations in ara-C metabolism remains to be elucidated. The present study aims to determine independent as well as the combined effect of ara-C metabolizing genes mRNA expression on in-vitro ara-C cytotoxicity and the role of FLT3-ITD and NPM mutations on mRNA expression of these genes. Diagnostic bone marrow sample (median blasts 65%; range 21 – 98%) from 98 adult patients with de novo AML (other than AML-M3) were included in this study. mRNA expression levels for each target gene relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. In vitro sensitivity or resistance was classified on the basis of the IC-50 values <6uM and >6uM ara-C respectively. FLT3 ITD and NPM mutation status at diagnosis were determined through PCR followed by Genescan analysis using genomic DNA samples. Type of NPM mutation was identified by sequencing. When ara-C IC-50 values were compared with the mRNA expression levels of these candidate genes, Ara-C sensitive samples (n= 30; IC-50 < 6uM) showed significantly higher mRNA expression of dCK and hENT1 compared to those with Ara-C resistance (n=51) IC50 >6uM (median 314 (61.56 – 1232) vs. 180 (31.87 – 749.2); p = 0.0004 and median 172.1 (44.12 – 657.6) vs. 96.19 (37.49 – 432.4), p= 0.0008 respectively. RRM1 and NT5C2 did not show any association with in vitro Ara-C cytotoxicity, while CDA showed a trend towards association with lower CDA expression in ara-C sensitive samples. Based on these findings we put forward Ara-C resistance index (RI). RI is calculated by the formula RI = ΔCT (dCK X ENT1)/ ΔCT CDA. (Smaller ΔCT value= higher mRNA expression). RI values were significantly higher in resistant (IC50 >6uM) compared to sensitive cells (median: 6.084; range 1.89–11.82) vs. 3.702 (1.89–9.80); p=<0.0001). This association should now be validated in an independent cohort. Effects of NPM and FLT3 mutation status on Ara-C metabolizing genes were then evaluated. No significant association was found between FLT3-ITD status and the mRNA expression of these candidate genes. Interestingly, dCK mRNA levels were significantly higher in samples with NPM mutation (n=39) compared to NPM wild type (n=59); median 272.3 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.01. When analysed separately, patients with NPM type A mutation (n=27) showed significantly higher dCK expression (median 347.4 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.003 compared to those with wild type NPM1. This first report showing an association between expression profiles of ara-C metabolizing genes and NPM mutation should form the basis for evaluating their clinical correlations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Oleic and palmitic acids induce hepatic angiopoietin-like 4 expression predominantly via PPAR-γ in Larimichthys crocea

2021 ◽  
pp. 1-27
Author(s):  
Xiaojun Xiang ◽  
Shangzhe Han ◽  
Dan Xu ◽  
Qiuchi Chen ◽  
Renlei Ji ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Mrna Expression ◽  
Regulatory Mechanisms ◽  
Large Yellow Croaker ◽  
Peroxisome Proliferator ◽  
Larimichthys Crocea ◽  
Peroxisome Proliferator Activated Receptor ◽  
Triacylglycerol Metabolism ◽  
Palmitic Acids

Abstract Angiopoietin-like 4 (ANGPTL4) is a potent regulator of triacylglycerol metabolism but knowledge of the mechanisms underlying ANGPTL4 transcription in response to fatty acids is still limited in teleost. In this study, we explored the molecular characterization of ANGPTL4 and regulatory mechanisms of ANGPTL4 in response to fatty acids in large yellow croaker (Larimichthys crocea). Here, croaker angptl4 contained a 1416 bp open reading frame encoding a protein of 471 amino acids with highly conserved 12-amino acid consensus motif. Angptl4 was widely expressed in croaker, with the highest expression in the liver. In vitro, oleic and palmitic acids (OA and PA) treatments strongly increased angptl4 mRNA expression in croaker hepatocytes. Moreover, angptl4 expression was positively regulated by peroxisome proliferator-activated receptor family (PPAR-α, β and γ) and expression of pparγ was also significantly increased in response to OA and PA. Moreover, inhibition of PPARγ abrogated OA or PA-induced angptl4 mRNA expression. Beyond that, PA might increase angptl4 expression partly via the insulin signaling. Overall, the expression of ANGPTL4 is strongly upregulated by OA and PA via PPARγ in the liver of croaker, which contributes to improve the understanding of the regulatory mechanisms of ANGPTL4 in fish.

scholarly journals Overexpression of lncRNA TUG1 Alleviates NLRP3 Inflammasome-Mediated Cardiomyocyte Pyroptosis Through Targeting the miR-186-5p/XIAP Axis in Coronary Microembolization-Induced Myocardial Damage

2021 ◽  
Vol 12 ◽  
Author(s):  
You Zhou ◽  
Tao Li ◽  
Zhiqing Chen ◽  
Junwen Huang ◽  
Zhenbai Qin ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Myocardial Injury ◽  
Culture Supernatant ◽  
Myocardial Damage ◽  
Luciferase Reporter ◽  
Cell Culture Supernatant ◽  
Coronary Microembolization ◽  
Lncrna Tug1

Coronary microembolization (CME) is a complicated problem that commonly arises in the context of coronary angioplasty. The lncRNA taurine-up regulated gene 1 (TUG1), significantly contributes to cardiovascular diseases; however, its contribution to CME-induced myocardial damage remains elusive. Herein, we establish the rat CME model and investigate the role of TUG1 in CME. The cell viability was evaluated via CCK-8 assay. Serum and cell culture supernatant samples were evaluated via ELISA. The dual luciferase reporter (DLR) assay, RIP, and RNA-pull down were conducted to validate the associations between TUG1 and miR-186-5p as well as miR-186-5p and XIAP. The expression of TUG1, miR-186-5p, and XIAP mRNA were determined by RT-qPCR, and proteins were evaluated via immuneblotting. As a result, TUG1 and XIAP were significantly down-regulated, and the miR-186-5p level was found to be remarkably up-regulated in CME myocardial tissues. Overexpression of TUG1 alleviated CME-induced myocardial injury and pyroptosis, whereas TUG1 knockdown showed the opposite effects. The DLR assay, RIP, and RNA-pull down results reveal that TUG1 directly targets miR-186-5p and miR-186-5p directly targets XIAP. In vitro rescue experiments show that TUG1 overexpression alleviates LPS-caused cardiomyocyte injury and pyroptosis via sponging miR-186-5p and regulating XIAP, and depression of miR-186-5p reduces LPS-induced cardiomyocyte injury and pyroptosis by targeting XIAP. Concludingly, the overexpression of TUG1 alleviates NLRP3 inflammasome-mediated cardiomyocyte pyroptosis through targeting the miR-186-5p/XIAP axis in CME-induced myocardial injury.

2′-Deoxy-5-Azacytidine Enhances Cytotoxic Effect Of Other Anti-Leukemic Agents Synergistically In Acute Lymphoblastic Leukemia Cell Line

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5564-5564
Author(s):  
Kimiyoshi Sakaguchi ◽  
Hiroyoshi Takahashi
Keyword(s):  
Mrna Expression ◽  
Mtt Assay ◽  
Research Funding ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Expression Levels ◽  
Apoptotic Genes ◽  
Mrna Expression Levels

Abstract Introduction Advances in chemotherapy have improved the outcome of childhood acute lymphoblastic leukemia (ALL). However, leukemia cells in refractory ALL are often resistant to anti-leukemic agents. Although recent studies have focused on the epigenetic changes in refractory leukemia, the relationship between the demethylating agent 2′-deoxy-5-azacytidine (decitabine, DAC) and ALL remains unclear. Here, we examine the combined effects of DAC and anti-leukemic agents such as clofarabine (CLO) and etoposide (ETO) on the ALL cell line CCRF-CEM. Methods and results In vitro drug sensitivity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. We cultured CCRF-CEM cells for 72 hours with or without DAC, and then removed DAC (when present) prior to culturing CCRF-CEM cells for 48 hours with ETO or CLO, or without chemotherapeutic drugs. After culturing for 48 hours, we removed the chemotherapeutic drugs and measured in vitro drug sensitivity using MTT assay. The MTT assay was performed in triplicate. We then evaluated the inhibitory concentration at 50% (IC50). IC50 for ETO, ETO+DAC, CLO, and CLO+DAC was 3.36, 0.625, 4.96, and 1.92, respectively. The combination Index (CI) was produced with Calcusyn® software, which uses the methodology of Chou and Talalay to perform formal synergy analyses. A CI < 1 indicated a synergistic effect. The CI was 0.026 for ETO+DAC and 0.431 for CLO+DAC. We assayed with Annexin-V, PI staining, and caspase-3/7 to detect apoptosis. We observed apoptosis rates of 31.6%, 53.3%, 31.2%, and 52.6% for ETO, ETO+DAC, CLO, and CLO+DAC, respectively. We observed greater caspase-3/7 activity with DAC+CLO and DAC+ETO than with CLO and ETO. Using real-time reverse transcription polymerase chain reaction (RT-qPCR) in CCRF-CEM cells, we examined mRNA expression levels for the pro-apoptotic genes BAK, BID, BAX, BAD, BIM, PUMA, ATM, TP53, and NOXA, as well as those for the anti-apoptotic genes BCL2, BCL2L1, and XIAP. The expression level of each target gene was calculated by normalizing it to the housekeeping gene GAPDH. The RT-qPCR was performed in triplicate. We used Student’s t test to compare the data. We observed DAC increased mRNA expression levels of BAX and NOXA, but decreased those for BAK, BID, PUMA, BCL2L1, ATM, TP53, and XIAP. We then analyzed the methylation status of pro- and anti-apoptotic genes after 48 hours incubation with or without DAC. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation with DAC was 1.3%, 3.3%, 2.5% and 72.9%, respectively. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation without DAC was 1.9%, 3.6%, 0.7% and 92.3%, respectively. There was no significant difference. Discussion Our results showed that DAC synergistically enhances CLO and ETO cytotoxicity, and this cytotoxic effect depends on caspase-3/7 activity. We examined mRNA expression levels of pro- and anti-apoptotic genes. We hypothesized that DAC would increase mRNA expression levels of most pro-apoptotic genes, and decrease mRNA levels of most anti-apoptotic genes. We found that DAC decreased some pro-apoptotic genes, such as BAK, BID, PUMA, ATM, and TP53, which disproves our hypothesis. Our present findings are similar to those of Shin et al., who reported that DAC decreased BID mRNA expression levels. However, they provided no explanation for this activity. Our results show that DAC did not demethylate the CpG of BAK, NOXA, BCL2L1, or XIAP. Thus, DAC must demethylate the CpG of other genes. Nevertheless, many genes are involved in apoptosis, and it remains unclear which genes are demethylated by DAC. Disclosures: Sakaguchi: Yakult Honsha Company: Research Funding; Japan Leukemia Research Fund: Research Funding; Japan Society for the Promotion of Science: Research Funding; Sanofi: Research Funding; Teijin Pharma: Research Funding.

83 BONE MORPHOGENETIC PROTEIN SIGNALING DURING INTERACTION OF THE BOVINE EMBRYO WITH OVIDUCTAL EPITHELIAL CELLS IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 149
Author(s):  
E. V. García ◽  
M. Hamdi ◽  
A. D. Barrera ◽  
M. J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Relative Abundance ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Expression Levels ◽  
Bmp Receptors ◽  
Bmp Signalling ◽  
Mrna Expression Levels

In previous studies, we have demonstrated that different signalling components of bone morphogenetic proteins (BMP) are expressed in an anatomically and temporally regulated fashion in the bovine oviduct. However, a local response of this signalling to the embryo presence has not been elucidated yet. The aim of the present study was to evaluate whether the interaction of the embryo with the oviduct can induce changes in the gene expression of BMP signalling components. For this purpose, we used an in vitro co-culture system of a bovine oviducal epithelial cell (BOEC) monolayer with pre-implantation embryos in 2 developmental time points: before and during the main phase of embryonic genome activation (EGA). Isthmus epithelial cells from post-ovulatory stage oviducts (Day 2–4) were cultured in 500 μL of SOF + 10% FCS in 4-well plates at 38.5°C, 5% CO2, 5% O2, and 90% N2. On Day 6 of culture, medium was replaced with SOF + 5% FCS, and 24 h later BOEC monolayer was cultured in the absence or presence of in vitro-produced embryos from 2- to 8-cell stage [G1 BOEC; 33–54 h post-insemination (hpi)] or from 8- to 16-cell stage (G2 BOEC; 54–98 hpi) in the same conditions. In both groups, a polyester mesh was used to define a local co-culture area, and 30 embryos per well were placed in a 6 × 5 grid over the monolayer. In addition, as control groups, embryos in both developmental stages were cultured either in SOF + 5% FCS (G1 FCS and G2 FCS) or in SOF + 3 mg mL−1 BSA (G1 BSA and G2 BSA). At 54 hpi (G1 BOEC/BSA/FCS) or 98 hpi (G2 BOEC/BSA/FCS), embryos that reached 8- or 16-cell stage, respectively, were transferred to SOF + BSA and cultured until Day 9. The mRNA expression levels of 3 BMP receptors (BMPRIA/IB/II), 2 signalling proteins (SMAD1/5), 1 inhibitor (SMAD6), and 1 target gene (ID2) were analysed by qPCR in 5 samples of BOEC cultured with or without embryos before or during EGA, and in 3 pools of 10 embryos at 8 (54 hpi), 16 (98 hpi), and blastocyst stage (Day 7–8) from all groups. Genes H2A.Z and ACTG1 were used as housekeeping genes, and statistical differences were assessed by ANOVA. The presence of the embryo, irrespective the stage, significantly reduced the expression levels of BMPRIB, BMPRII, SMAD1, SMAD6, and ID2 in BOEC. Embryos that interacted with BOEC before EGA (G1 BOEC) showed a significant increase in the relative abundance of SMAD1 at the 8-cell stage compared with controls. Moreover, embryos that interacted with BOEC during EGA (G2 BOEC) showed a significant increase in the relative abundance of BMPRIB, BMPRII, and ID2 at the 16-cell stage when compared with controls. However, no differences were observed in the mRNA expression levels of BMP signalling components in the blastocysts between groups. In conclusion, local embryo-oviduct interaction in vitro induces changes in the transcriptional levels of BMP signalling, causing a bidirectional response that reduces the expression levels of this signalling in the oviducal cells while increases them in the embryo at early stages. This suggests that BMP signalling pathway could be involved in an early cross-talk between the bovine embryo and the oviduct during first stages of development.

A Clinical Study of Bone Marrow Stromal Cell Therapy in Patients with Refractory Cgvhd by Down-Regulating the Jagged2 Gene

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4673-4673
Author(s):  
Jianyu Weng ◽  
Xin Huang ◽  
Suxia Geng ◽  
Chengwei Luo ◽  
Suijing Wu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Control Group ◽  
Expression Levels ◽  
Mrna Expression Levels

Abstract Abstract 4673 Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. Bone Marrow Stromal Cells (MSCs) are involved in tissue repair and modulating immune responses in vitro and in vivo. MSCs as salvage treatment for refractory cGVHD have been reported in our previous study, however, the possible mechanism have yet not to be determined. Between November 2006 and November 2010, 18 patients were diagnosed with refractory cGVHD, 8 patients were treated with in vitro expanded BM-derived MSCs as a compassionate treatment for refractory cGVHD, 10 patients that did not receive BMSCs treatment were control group. The median MSC dose given was 0.6×106/kg body weight. MSCs were harvested fresh from culture and administered to the patients by intravenous infusions over 30 minutes. The median time of MSC administrations was 3 (range, 2–6). The response was assessed monthly after BMSCs treatment, and the total follow-up period was 6 months. The organ response and the overall response were used to determine the therapeutic efficacies of MSC for refractory cGVHD. The expression of the Jagged2 gene of peripheral blood mononuclear cells in patients at the assessment points were analyzed using the TaqMan real-time polymerase chain reaction, with ABL mRNA expression levels as an internal reference. After BMSCs treatment, a total of 6 patients (75%) had an overall response (PR n=6), and 2 patients had a minor partial response (mPR n=2). The expression levels of Jagged2 mRNA in these cases at the diagnosis of refractory cGVHD were significantly increased, compared with none cGVHD patients (23.94%±18.68% vs 3.76%±1.50%, P < 0.05), and the copies of Jagged2 mRNA in BMSC treatment responsed patients' peripheral blood were significantly reduced (5.15%±3.25%, P <0.05), while Jagged2 mRNA expression levels of the control group were no significant difference (P> 0.05). Our pilot study showed that Jagged2 gene reproduction upregulated when the cGVHD is active, so, dynamic monitoring of Jagged2 mRNA expression may have the potential effect on predicting the activity of chronic graft-versus-host disease. Mechanism of Bone marrow stromal cells to treat refractory cGVHD may be related to down-regulation of donor T cells Notch ligand Jagged2 gene expression, which suppression of T cell Notch signaling pathway activation, thus inducing immune tolerance. Disclosures: No relevant conflicts of interest to declare.

Relationship between plasma BRCA1 mRNA expression and sensitivity to docetaxel and cisplatin in gastric cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14509-e14509
Author(s):  
Jie Shen ◽  
Jia Wei ◽  
Wen xian Guan ◽  
Hao Wang ◽  
Yi tao Ding ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Mrna Expression ◽  
Predictive Biomarker ◽  
Mrna Levels ◽  
Expression Levels ◽  
In Vitro Chemosensitivity ◽  
Brca1 Mrna ◽  
Cisplatin Sensitivity ◽  
Mrna Expression Levels

e14509 Background: Tumor-derived RNA species successfully detected in plasma may have potential for use in disease and treatment assessment. Although BRCA1 mRNA expression levels in tumor are associated with cisplatin sensitivity but docetaxel resistance, the role of plasma BRCA1 mRNA as a predictive biomarker remains to be known. The aim of this study was to investigate the association between plasma BRCA1 mRNA expression and in vitro chemosensitivity to docetaxel and cisplatin in gastric cancer. Methods: 150 freshly-removed gastric tumor specimens and corresponding blood samples before surgery were collected. Docetaxel and cisplatin sensitivity was determined by histoculture drug response assay (HDRA) procedures. Plasma and tumor BRCA1 mRNA expression levels were determined by quantitative RT-PCR. Results: A significant correlation was observed between plasma and tumor BRCA1 mRNA expression levels (rho=0.558, P<0.001). Plasma BRCA1 mRNA expression level was positively correlated with in vitro sensitivity to docetaxel (docetaxel-sensitive sub-group: 1.25, 95% CI: 1.04-1.47; docetaxel-resistant sub-group: 0.50, 95% CI: 0.23-0.78; p<0.001) but negatively correlated with sensitivity to cisplatin in gastric cancer (cisplatin-sensitive sub-group: 0.84, 95% CI: 0.61-1.08; cisplatin-resistant sub-group:1.20, 95% CI: 0.84-1.56; p=0.083). There was no significant association between clinical characteristics and plasma BRCA1 mRNA levels or in vitro chemosensitivity. Conclusions: It was demonstrated for the first time that plasma BRCA1 mRNA expression was associated with in vitro chemosensitivity to docetaxel and cisplatin, which provided preliminary evidence for using plasma mRNA expression as an approach to predict response to docetaxel or cisplatin based chemotherapy in the clinic.

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