scholarly journals Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhifu Cui ◽  
Zhichao Zhang ◽  
Felix Kwame Amevor ◽  
Xiaxia Du ◽  
Liang Li ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Expression Pattern ◽  
Epithelial Transport ◽  
Egg Laying ◽  
Luciferase Reporter ◽  
Biological Clocks ◽  
Luciferase Reporter Gene ◽  
Intracellular Ca ◽  
Tubular Gland ◽  
Mrna And Protein Expression

Abstract Background miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. Results We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport. Conclusions Clock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification.

scholarly journals Circadian miR-449c-5p Regulates Uterine Ca2+ Transport During Eggshell Calcification in Chickens

2021 ◽  
Author(s):  
Zhifu Cui ◽  
Zhichao Zhang ◽  
Felix Kwame Amevor ◽  
Xiaxia Du ◽  
Liang Li ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Expression Pattern ◽  
Epithelial Transport ◽  
Clock Genes ◽  
Biological Clock ◽  
Egg Laying ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Intracellular Ca ◽  
Tubular Gland

Abstract Background: miRNAs regulate circadian patterns by modulating animal biological clock. Clock genes exhibited a cosine expression pattern in the fallopian tube of chicken uterus in our previous study. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in chicken uterus and, if so, how they regulate egg-laying rhythms are not clear. Here, we selected 18 layer hens with similar ovipositional rhythmicity (three birds were sacrificed for study per at 4 h intervals throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens.Results: We identified six circadian miRNAs mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. miR-449c-5p showed an opposite expression profile with ATP2B4 within a 24h cycle in chicken uterus; it inhibited mRNA and protein expressions of ATP2B4 in uterine tubular gland cells. Additionally, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. Similar results were found after ATP2B4 knockdown by miR-449c-5p. These results indicated that ATP2B4 promoted uterine Ca2+ trans-epithelial transport. Conclusions: Clock-controlled miR-449c-5p regulates Ca2+ transport in chicken uterus by targeting ATP2B4 during eggshell calcification.

scholarly journals Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens

2021 ◽  
Author(s):  
Zhifu Cui ◽  
Zhichao Zhang ◽  
Felix Kwame Amevor ◽  
Xiaxia Du ◽  
Liang Li ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Expression Pattern ◽  
Epithelial Transport ◽  
Clock Genes ◽  
Biological Clock ◽  
Egg Laying ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Tubular Gland ◽  
Circadian Patterns

Abstract Background: miRNAs regulate circadian patterns by modulating animal biological clock. Clock genes exhibited a cosine expression pattern in the fallopian tube of chicken uterus in our previous study. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in chicken uterus and, if so, how they regulate egg-laying rhythms are not clear. Here, we selected 18 layer hens with similar ovipositional rhythmicity (three birds were sacrificed for study per at 4 h intervals throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. Results: We identified six circadian miRNAs mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. miR-449c-5p showed an opposite expression profile with ATP2B4 within a 24h cycle in chicken uterus; it inhibited mRNA and protein expressions of ATP2B4 in uterine tubular gland cells. Additionally, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. Similar results were found after ATP2B4 knockdown by miR-449c-5p. These results indicated that ATP2B4 promoted uterine Ca2+ trans-epithelial transport. Conclusions: Clock-controlled miR-449c-5p regulates Ca2+ transport in chicken uterus by targeting ATP2B4 during eggshell calcification.

Urea-associated oxidative stress and Gadd153/CHOP induction

1999 ◽  
Vol 276 (5) ◽  
pp. F786-F793 ◽  
Author(s):  
Zheng Zhang ◽  
Xiao-Yan Yang ◽  
David M. Cohen
Keyword(s):  
Oxidative Stress ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Kidney Medulla ◽  
Urea Treatment ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Mrna And Protein Expression ◽  
Reporter Gene Analysis

Urea treatment (100–300 mM) increased expression of the oxidative stress-responsive transcription factor, Gadd153/CHOP, at the mRNA and protein levels (at ≥4 h) in renal medullary mIMCD3 cells in culture, whereas other solutes did not. Expression of the related protein, CCAAT/enhancer-binding protein (C/EBP-β), was not affected, nor was expression of the sensor of endoplasmic reticulum stress, grp78. Urea modestly increased Gadd153 transcription by reporter gene analysis but failed to influence Gadd153 mRNA stability. Importantly, upregulation of Gadd153 mRNA and protein expression by urea was antioxidant sensitive. Accordingly, urea treatment was associated with oxidative stress, as quantitated by intracellular reduced glutathione content in mIMCD3 cells. In addition, antioxidant treatment partially inhibited the ability of urea to activate transcription of an Egr-1 luciferase reporter gene. Therefore oxidative stress represents a novel solute-signaling pathway in the kidney medulla and, potentially, in other tissues.

scholarly journals Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca2+-dependent Pathways in Fission Yeast

2006 ◽  
Vol 17 (11) ◽  
pp. 4790-4800 ◽  
Author(s):  
Lu Deng ◽  
Reiko Sugiura ◽  
Mai Takeuchi ◽  
Masahiro Suzuki ◽  
Hidemine Ebina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Tandem Repeats ◽  
Living Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Concentration Dependent Manner ◽  
Intracellular Ca ◽  
Calcineurin Activity

In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1+ gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl2 caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Δpmc1, the response was markedly enhanced, reflecting its high intracellular Ca2+. Agents expected to induce Ca2+ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall–damaging agents. However, knockout of yam8+ or cch1+ did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca2+. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx, but it was not required for the stimulation by elevated extracellular Ca2+, suggesting two distinct pathways for calcineurin activation.

Different Induction Effects of Praziquantel Racemate and Enantiomers on the Enzyme CYP3A4 Mediated by Pregnane X Receptor and Its Variants

2021 ◽  
Vol 21 ◽  
Author(s):  
Ran Meng ◽  
Xueli Zhang ◽  
Haina Wang ◽  
Danlu Zhang ◽  
Xin Zhao
Keyword(s):  
Hepg2 Cells ◽  
Pregnane X Receptor ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Concentration Dependent Manner ◽  
Protein Levels ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Expression Plasmids

Background: Praziquantel (PZQ), which possesses an asymmetric center, is classified as a pyrazinoisoquinoline and has been the mainstay in the treatment of schistosomiasis since 1980. PZQ undergoes a pronounced first-pass metabolism in the liver through the CYP450 system which could be mediated by nuclear receptors. Objective: The purpose of this study was to investigate the possible different induction effects of CYP3A4 by PZQ racemate and enantiomers via the pregnane X receptor (PXR) and the effect of PXR polymorphism on the induction potency of PZQs. Methods: The dual-luciferase reporter gene systems constructed in HepG2 cells were used to measure the abilities of PZQs to induce CYP3A4 expression mediated by PXR. The mRNA and protein levels of CYP3A4 were evaluated by polymerase chain reaction (PCR) and western blotting, respectively. Results: In HepG2 cells transfected with PXRwt, PXR158, PXR163, PXR370 or PXR403 expression plasmids, PZQ racemate and its enantiomers up-regulated the luciferase activity in a concentration-dependent manner, while reaching saturation after transfected with PXR379 expression plasmids. The mRNA and protein expression of CYP3A4 was effectively activated in PXR-transfected HepG2 cells. The induction ability of CYP3A4 mediated by PXR activation by PZQ racemate and its enantiomers were statistically different between the same PXR group and different PXR groups. Conclusion: The enantioselective induction effects of PZQs on CYP3A4 were related to the enantioselective activations of PXR by PZQs and were influenced by the PXR gene polymorphism. These findings provide a basis for further understanding the enantiomeric metabolism and the variable efficacy of PZQs.

scholarly journals miR-99a-3p Targeting EIF4EBP1 Affects B Lymphocytes Function Through Autophagy and Aggravates SLE Disease Progression

2021 ◽  
Author(s):  
meng yang ◽  
Deng Danqi
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Protein Expression ◽  
Reporter Gene ◽  
Immune Cells ◽  
Target Genes ◽  
Kidney Tissue ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mrna And Protein Expression

Abstract Background:Overactivation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNA is a research hotspot.In this study, the second-generation high-throughput sequencing found that the expression of miR-99a-3p in SLE decreased, but the specific mechanism is still unclear.The purpose of this study is to explore the potential target genes, target cells of miR-99a-3p and their potential mechanisms affecting the progression of SLE.Methods: Isolate PBMC from healthy individulas and SLE patients, transfect Ball-1, Jurkat, THP-1 and K562 cells with miR-99a-3p agomir and antagomir,detect miR-99a-3p, predict target genes and autophagy pathway mRNA and protein expression by RT-qPCR and Western blotting.CCK-8 method detects cell proliferation, PI method detects cell cycle, flow cytometry detects cell apoptosis, and luciferase reporter gene experiment determines miR-99a-3p target genes.With C57BL/6J mice as control,construct miR-99a-3p overexpression and interference model based on MRL/lpr mice,ELISA detects plasma ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS.Pathological analysis of HE staining and C3 immunofluorescence(IF) deposition in mouse kidney tissue,Immunohistochemistry(IHC) detects changes in target genes and pathway proteins in kidney tissue,isolate B cells to verify the differential expression of miR-99a-3p, target genes, pathway mRNA and protein.Results: Compared with healthy individulas, miR-99a-3p in SLE was down-regulated, while EIF4EBP1, LC3II, LAMP-2A mRNA and protein expression were up-regulated.After Ball-1 was transfected with miR-99a-3p agomir, cell proliferation decreased and apoptosis increased.After transfected with miR-99a-3p antagomir, the effect was opposite;Luciferase reporter gene detection proved that miR-99a-3p directly targets EIF4EBP1. Rescue experiments confirmed the interaction model between miR-99a-3p and EIF4EBP1. Clinical, in vitro, and in vivo experiments further confirmed that miR-99a-3p agomir can reduce the expression of EIF4EBP1, LC3-Ⅱ, and LAMP-2A, while miR-99a-3p antagomir had the opposite effect.In vivo experiment antagomir group mice serum ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS were higher than those in the MRL/lpr group, EIF4EBP1, LC3-Ⅱ, LAMP-2A mRNA, protein and IHC levels also increased, and the urinary protein and C3 IF deposition of mice in the antagomir group were increased, and the related indexes of mice in the agomir group were lower than those in the MRL/lpr group.Conclusion:The expression of miR-99a-3p in SLE PBMC was down-regulated. Up-regulation of miR-99a-3p by transfection can protect B cells from autophagy mediated by EIF4EBP1.The down-regulation of miR-99a-3p induces autophagy by regulating the autophagy signaling pathway mediated by EIF4EBP1 in SLE B cells. These results indicate that miR-99a-3p and EIF4EBP1 may be potential targets of SLE.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma

2020 ◽  
Vol 23 ◽  
Author(s):  
Zheng Dong ◽  
Qing-Hua Xu ◽  
Yuan-Bin Zhu ◽  
Yong-Feng Wang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Lung Squamous Cell Carcinoma ◽  
Luciferase Reporter ◽  
Control Group ◽  
Vascular Endothelial ◽  
Luciferase Reporter Gene ◽  
Endothelial Growth Factor

Aims : The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). Methods: A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment was selected. The expression of miR-22 was detected by fluorescence quantitative real-time PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGF-D) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. Results: The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 cotransfection group reversed the changes. Conclusion: We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

2020 ◽  
Vol 20 (6) ◽  
pp. 715-723
Author(s):  
Natarajan Nandakumar ◽  
Pushparathinam Gopinath ◽  
Jacob Gopas ◽  
Kannoth M. Muraleedharan
Keyword(s):  
Synergistic Effect ◽  
Hodgkin’S Lymphoma ◽  
A549 Cells ◽  
Hodgkin's Lymphoma ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lymphoma Cells ◽  
Nuclear Extracts ◽  
Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

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