scholarly journals A Potential miRNA-mRNA Network for Dementia and Hernia Crosstalk

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
De-jian Chen ◽  
Da-peng Li
Keyword(s):  
Reporter Gene ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Clinical Samples ◽  
Luciferase Reporter Gene ◽  
Tissue Samples ◽  
Regulatory Pathways ◽  
Gene Assay ◽  
Potential Link

Background. It has been reported that there may be a potential link between hernia and dementia. However, the exact mechanisms of their association have not been established. This study is aimed at constructing miRNA-mRNA networks to elucidate on the potential link between dementia and hernia. Methods. Gene expression profiles for dementia, herniation, and skeletal muscle were downloaded from the GEO database after which differentially expressed mRNAs and miRNAs were obtained. In addition, fascia tissue samples were obtained during surgery. A total of 41 patients were recruited in this study, and expression levels of candidate genes were examined using quantitative RT-PCR. Luciferase reporter gene assays were used to identify potential miRNA-mRNA regulatory pathways. Results. Differentially expressed mRNAs and miRNAs were screened. A potential miRNA-mRNA network revealing the crosstalk mechanism between herniation and dementia was identified. Single cell analysis revealed that PI16 was highly enriched in adipose tissues, skeletal muscles, and in the skin. GSEA enrichment analysis showed that PI16 is involved in adipose metabolism, muscle functions, and energy metabolism. In clinical samples, PI16 was found to be upregulated in hernia, while miR-4451 was found to be downregulated. The luciferase reporter gene assay revealed that downregulation of circulating miR-4451 may be responsible for the upregulated PI16 expression in hernia sacs. Conclusions. We constructed an miRNA-mRNA network that shows the potential association between dementia and hernia. We also found that miR-4451 regulates the PI16 expression, which may be a key target and biomarker for hernia pathogenesis and dementia crosstalk.

scholarly journals Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2585 ◽  
Author(s):  
Lehao Wu ◽  
Weiyue Zhang ◽  
Xin Qiu ◽  
Chaoran Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
D1 Receptor ◽  
Assay System ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Antagonistic Effects ◽  
Gene Assay

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

scholarly journals eNOS Promoter Activation by Red Wine Polyphenols: an Interaction Study

2013 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
Author(s):  
E. Kurin ◽  
N. Fakhrudin ◽  
M. Nagy
Keyword(s):  
Reporter Gene ◽  
Red Wine ◽  
Interaction Analysis ◽  
Mass Action ◽  
Luciferase Reporter ◽  
Interaction Study ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Wine Polyphenols ◽  
Red Wine Polyphenols

Beneficial effects of red wine polyphenols on cardiovascular health are well known. The aim of our research was an interaction study of four red wine polyphenols – resveratrol (R), quercetin (Q), kaempferol (KF) and isorhamnetin (IR) of their ability to activate endothelial NO synthase (eNOS) promoter when used alone and in equimolar mixtures. To determine their activity, we performed a luciferase reporter gene assay on EA.hy926 cells stably transfected with a luciferase reporter gene construct containing eNOS promoter. The Bradford assay was also performed to account the cytotoxicity and/or the cell number differences. The median effect equation, as an interaction analysis evaluating synergy or antagonism of the combinations was done according to mass-action law principle. Isobolographic method was performed on selected double mixtures and dose reduction index was calculated for all mixtures. All single polyphenols activated eNOS promoter. The EC50 values were in micromolar concentrations ranging from 3.44 μM (R2 = 0.96) for kaempferol to 9.89 μM for isorhamnetin (R2 = 0.94). All mixtures activated eNOS promoter, but their interactions varied from synergy (Q+R, Q+IR+KF, Q+R+KF and Q+R+IR+KF), through additive (R+IR+KF) to antagonistic interaction (R+IR, R+KF, Q+IR, Q+KF, IR+KF and R+Q+IR). In this study, we show for the first time that red wine polyphenols activated eNOS promoter when used alone and in mixtures with different type of interactions.

lncRNA KCNQ1OT1 promotes the proliferation, migration and invasion of retinoblastoma cells by upregulating HIF-1α via sponging miR-153-3p

2020 ◽  
Vol 68 (8) ◽  
pp. 1349-1356
Author(s):  
Yujin Wang ◽  
Jixiang Wang ◽  
Hongyan Hao ◽  
Xiangxia Luo
Keyword(s):  
Colony Formation ◽  
Rna Binding ◽  
Hypoxia Inducible Factor ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Tissue Samples ◽  
Qrt Pcr ◽  
Gene Assay

It is reported that lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) is oncogenic in many cancers. This work aimed at probing into its expression and biological functions in retinoblastoma (RB) as well as its regulatory effects on miR-153-3p and hypoxia-inducible factor-1α (HIF-1α). In our study, RB samples in pair were collected, and quantitative real-time PCR (qRT-PCR) was employed for examining the expression levels of KCNQ1OT1, miR-153-3p and HIF-1α. KCNQ1OT1 short hairpin RNAs were transfected into SO-Rb50 and HXO-RB44 cell to inhibit the expression of KCNQ1OT1. The proliferative activity, colony formation ability and apoptosis were examined through cell counting kit-8 assay, colony formation assays, Transwell assay and flow cytometry, respectively. qRT-PCR and western blot analysis were used for analyzing the changes of miR-153-3p and HIF-1α induced by KCNQ1OT1. The regulatory relationships between miR-153-3p and KCNQ1OT1, miR-153-3p and HIF-1α were examined by dual luciferase reporter gene assay and RNA-binding protein immunoprecipitation assay. The results of our study showed that KCNQ1OT1 expression was markedly enhanced in RB tissue samples, and KCNQ1OT1 knockdown had an inhibitory effect on the proliferation, migration, invasion and viability of RB cells. There were two validated binding sties between KCNQ1OT1 and miR-153-3p, and KCNQ1OT1 negatively regulated the expression of miR-153-3p in RB cells. HIF-1α was a target gene of miR-153-3p, and could be positively regulated by KCNQ1OT1. In conclusion, our study indicates that KCNQ1OT1 can increase the malignancy of RB cells via regulating miR-153-3p/HIF-1α axis.

scholarly journals Oxymatrine inhibits proliferation and migration of breast cancer cells by inhibiting miRNA-188 and upregulating its target gene, PTEN

2021 ◽  
Vol 20 (11) ◽  
pp. 2267-2272
Author(s):  
Xiaoying Ma ◽  
Zijiang Sang ◽  
Qinghua Zhang ◽  
Wenbiao Ma
Keyword(s):  
Breast Cancer ◽  
Reporter Gene ◽  
Target Gene ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Pten Gene ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Regulatory Loop

Purpose: To explore the potential biological functions of oxymatrine on breast cancer (BCa) cells and the underlying molecular mechanism.Methods: Relative levels of microRNA-188 (miRNA-188) and PTEN (gene of phosphate and tension homology deleted on chromosome ten) in BCa cells, MDA-MB-231 and TB549, were determined. The influence of oxymatrine treatment, miRNA-188 and PTEN on proliferative and migratory abilities in BCa cells were assessed by 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell counting kit-8 (CCK-8) and Transwell assay, respectively. The binding relationship between miRNA-188 and PTEN was evaluated by dual-luciferase reporter gene assay.Results: Oxymatrine downregulated miRNA-188 and upregulated PTEN in BCa cells. Proliferative and migratory activities in BCa were inhibited by treatment of oxymatrine (p < 0.05). Dual-luciferase reporter gene assay results indicated that PTEN was the target gene of miRNA-188. Furthermore, rescue experiments demonstrated that the regulatory loop, oxymatrine/miRNA-188/PTEN, was involved in the regulation of the migration and proliferation of BCa.Conclusion: Oxymatrine treatment inhibits BCa progression by downregulating miRNA-188, leading to the upregulation of PTEN. The results of the current study may provide new insight into the diagnosis and treatment of BCa.

scholarly journals Circ_0000005 Facilitates Proliferation, Apoptosis, Migration and Invasion of Acute Myeloid Leukemia Cells via Modulating miR-139-5p/Tspan3 Axis

2020 ◽  
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Changcheng Zheng ◽  
Xiaoyu Zhu ◽  
Xiang Wan ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Abstract Background: Accumulating circular RNAs (circRNAs) are reported to be abnormally expressed in diverse cancers, hematologic malignancies included. This study aimed to investigate the biological function and underlying mechanisms of circ_0000005 in acute myeloid leukemia (AML).Materials and methods: Bone marrow samples were enrolled from AML patients with normal samples as controls. Circ_0000005, miR-139-5p and tetraspanin 3 (Tspan3) were detected by qRT-PCR and Western blot, respectively. AML cell lines (KG1 and HL60) were used as cell models. CCK-8, Transwell and flow cytometry assays were adopted to study the biological functions of circ_0000005 on AML cells in vitro. The interrelation between circ_0000005 and miR-139-5p was detected by bioinformatics, qRT-PCR, luciferase reporter gene assay, RNA pull-down assay, and RNA-binding protein immunoprecipitation (RIP) assays. Ultimately, Western blot, qRT-PCR, luciferase reporter gene assay were adopted to corroborate the interrelation between miR-139-5p and its target Tspan3. Results: Circ_0000005 was demonstrably elevated in both AML clinical samples and cell lines. Circ_0000005 overexpression promoted the viability, migration and invasion of AML cells, and repressed the apoptosis of AML cells, while silencing circ_0000005 showed opposite biological effects. Circ_0000005 interacted with miR-139-5p and repressed its expression, and Tspan3 was proved to be negatively regulated by miR-139-5p. Circ_0000005 could promote the expression of Tspan3 via repressing miR-139-5p, and the oncogenic functions of circ_0000005 were dependent on its regulatory function on miR-139-5p/Tspan3 axis.Conclusion: Circ_0000005 facilitates the malignant phenotypes of AML cells via miR-139-5p/Tspan3 axis. Circ_0000005 may serve as a potential therapeutic target in AML.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Gerontology ◽  
2022 ◽  
pp. 1-11
Author(s):  
Chengyuan Zhang ◽  
Ye Lu ◽  
Feng Yuan ◽  
Shilin Jiang
Keyword(s):  
Reporter Gene ◽  
Bioinformatics Analysis ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Tnf Α ◽  
Gene Assay ◽  
Sirt3 Expression

<b><i>Objective:</i></b> CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. <b><i>Methods:</i></b> The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. <b><i>Results:</i></b> It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. <b><i>Conclusion:</i></b> CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

scholarly journals Development of a Functional Reporter Gene HTS Assay for the Identification of mGluR7 Modulators

2000 ◽  
Vol 5 (4) ◽  
pp. 255-261 ◽  
Author(s):  
G. C. Terstappen ◽  
A. Giacometti ◽  
E. Ballini ◽  
L. Aldegheri
Keyword(s):  
Reporter Gene ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Signal Transduction Pathway ◽  
Resistant Cell ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Camp Levels

For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC50 of 0-9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.

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