scholarly journals MiR-545-3p Upregulated in Osteoporosis and Regulates The Apoptosis of Osteoblasts by Targeting SIRT6

2020 ◽  
Author(s):  
Qi-Ming Ma ◽  
Xiao-Jing Li ◽  
Shao-Bao Pei ◽  
Bo-Wen Li ◽  
Guo-Song Han ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Reporter Gene ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Osteogenic Medium ◽  
Luciferase Reporter Gene ◽  
Transfected Cells ◽  
Gene Assay ◽  
Related Proteins

Abstract Background: The imbalance of proliferation and apoptosis plays an important role in the pathogenesis of osteoporosis. MicroRNAs play an important role in the apoptosis of osteoblasts cells. However, the role and potential mechanism of miR-545-3p in regulating osteoblast apoptosis remain unclear. The purpose of this study was to investigate the effect of miR-545-3p on osteoblast cells apoptosis and explore the mechanism of osteoporosis.Methods: Osteogenic medium was used to induce the differentiation of osteoblasts MC3T3-E1 to construct the osteoporosis model, and the expression of ALP, Runx2, OCN were detected by western blot; miR-545-3p mRNA was detected by RT-PCR. Transfected with miR-545-3p mimics into MC3T3-E1, then flow cytometry was used to detected the changes of apoptosis status. The expression of apoptosis related proteins Bcl-2 and Bax were detected by western blot. Bioinformatics was used to analyze the binding protein of miR-545-3p, and luciferase reporter gene experiment was used to verify whether miR-545-3p directly targets SIRT6; RT-qPCR and western blot were used to detect the expression level of SIRT6 after transfection of miR-545-3p mimics or miR-545-3p inhibitor. After co-transfection of miR-545-3p mimics and pcDNA3.1-SIRT6, the apoptosis status of osteoblasts was analyzed by flow cytometry, and the expression of apoptosis related proteins Bcl-2 and Bax were detected by western blot.Results: The expression of miR-545-3p in patients with osteoporosis was significantly higher than that of normal in GEO database (P<0.05). After osteoblasts were cultured in osteogenic medium, the expression of ALP, Runx2 and OCN was increased, and the expression of miR-545-3p was decreased. Flow cytometry analysis showed that overexpression of miR-545-3p promoted the apoptosis of osteoblasts. Western blot results showed that overexpression of miR-545-3p promoted the expression of Bax and decreased the expression of Bcl-2. Bioinformatics analysis showed that miR-545-3p could target SIRT6. The results of real-time PCR and western blot showed that SIRT6 expression was significantly inhibited by miR-545-3p mimics (P<0.05). Luciferase reporter gene assay showed that miR-545-3p significantly inhibited luciferase activity of wild-type SIRT6-3'UTR plasmid transfected cells (P<0.05), but had no effect on luciferase activity of mutant SIRT6-3'UTR plasmid transfected cells (P<0.05). However, co-transfection of miR-545-3p mimics and pcDNA3.1-SIRT6 could reduce the apoptosis, and western blot results showed that co-transfection promoted the expression of Bcl-2 and decreased the expression of Bax.Conclusions: miR-545-3p can promote the apoptosis of osteoblasts by inhibiting the expression of SIRT6, which provides a certain idea for the treatment of osteoporosis.

scholarly journals The Differential Expression of miRNAs and a Preliminary Study on the Mechanism of miR-194-3p in Keloids

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Zhishan Xu ◽  
Bingyu Guo ◽  
Peng Chang ◽  
Qiang Hui ◽  
Wei Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Differential Expression ◽  
Real Time Pcr ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cultured Fibroblasts ◽  
Gene Assay ◽  
And Migration ◽  
Related Proteins

The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids.

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.

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.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

scholarly journals Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice

2004 ◽  
Vol 32 (3) ◽  
pp. 689-701 ◽  
Author(s):  
JG Lemmen ◽  
RJ Arends ◽  
AL van Boxtel ◽  
PT van der Saag ◽  
B van der Burg
Keyword(s):  
Reporter Gene ◽  
Imaging System ◽  
Luciferase Activity ◽  
Estrogenic Activity ◽  
Receptor Activation ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
In Vivo Model ◽  
Luciferase Reporter Gene

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

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.

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