scholarly journals MKL-1 regulates the stem cell marker. CD44 in breast cancer cells

2019 ◽  
Vol 78 ◽  
pp. 01002
Author(s):  
Zhou-Tong Dai ◽  
Ao Yao ◽  
Yuan Xiang ◽  
Jia Peng Li ◽  
Wei Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Malignant Tumors ◽  
Regulation Of Gene Expression ◽  
Gene Promoter ◽  
Expression Regulation ◽  
Stem Cell Marker ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Luciferase Reporter Gene ◽  
Factors Affecting

CD44, cluster of differentiation 44 is a typical marker of stem cells. At present, it has been found that CD44 is prevalent in various human malignant tumors, but its expression regulation mechanism is still not clear. The initiation of gene expression, the modification of RNA levels, and the regulation of protein levels are the main factors affecting the expression level of genes, and the most critical one is the regulation of gene expression by signaling pathways. Up to now, there has been no report on the role of MKL-1 in the cloning of the cd44 promoter. Therefore, this study intends to clone the cd44 gene promoter, construct its luciferase reporter gene vector, transfect the MKL-1 overexpression vector, and analyze how it affects transcriptional activity, in order to further study the expression regulation of cd44. The mechanism provides a powerful tool in the future.

scholarly journals Analyzing the whole-transcriptome profiles of ncRNAs and predicting the competing endogenous RNA networks in cervical cancer cell lines with cisplatin resistance

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Huimin Lv ◽  
Shanshan Jin ◽  
Binbin Zou ◽  
Yuxiang Liang ◽  
Jun Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cervical Cancer ◽  
Reporter Gene ◽  
Biological Function ◽  
Malignant Tumors ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Luciferase Reporter Gene ◽  
Network Analyses ◽  
Whole Transcriptome

Abstract Background Cervical cancer (CC) is one of the most common malignant tumors in women. In order to identify the functional roles and the interaction between mRNA and non-coding RNA (ncRNA, including lncRNA, circRNA and miRNA) in CC cisplatin (DDP) resistance, the transcription profile analysis was performed and a RNA regulatory model of CC DDP resistance was proposed. Methods In this study, whole-transcriptome sequencing analysis was conducted to study the ncRNA and mRNA profiles of parental SiHa cells and DDP resistant SiHa/DDP cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed for pathway analysis based on the selected genes with significant differences in expression. Subsequently, ceRNA network analyses were conducted using the drug resistance-related genes and signal-transduction pathways by Cytoscape software. Furthermore, a ceRNA regulatory pathway, namely lncRNA-AC010198.2/hsa-miR-34b-3p/STC2, was selected by RT-qPCR validation and literature searching. Further validation was done by both dual-luciferase reporter gene assays and RNA pull-down assays. Besides that, the changes in gene expression and biological function were further studied by performing si-AC010198.2 transfection and DDP resistance analyses in the SiHa and SiHa/DDP cells, respectively. Results Using bioinformatics and dual-luciferase reporter gene analyses, we found that AC010198.2/miR-34b-3p/STC2 may be a key pathway for DDP resistance in CC cells. Significant differences in both downstream gene expression and the biological function assays including colony formation, migration efficiency and cell apoptosis were identified in AC010198.2 knockdown cells. Conclusions Our study will not only provide new markers and potential mechanism models for CC DDP resistance, but also discover novel targets for attenuating it.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

scholarly journals Calcium Dynamics and Resting Transcriptional Activity Regulates Prolactin Gene Expression

Endocrinology ◽  
2002 ◽  
Vol 143 (9) ◽  
pp. 3548-3554 ◽  
Author(s):  
Carlos Villalobos ◽  
Lucía Núñez ◽  
William J. Faught ◽  
David C. Leaumont ◽  
Fredric R. Boockfor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Luciferase Reporter Gene ◽  
Transcriptional Responses ◽  
Prolactin Gene ◽  
Before And After ◽  
Single Living

Abstract Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

scholarly journals The 5′ Untranslated Region of the Capsid Protein 2 Gene of Mink Enteritis Virus Is Essential for Its Expression

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Shuang-Shuang Yang ◽  
Jigui Wang ◽  
Zhaoda Li ◽  
Shangjin Cui ◽  
Weiquan Liu
Keyword(s):  
Gene Expression ◽  
Capsid Protein ◽  
Gene Expression Regulation ◽  
Expression Regulation ◽  
Capsid Gene ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Vp2 Gene ◽  
Mink Enteritis Virus ◽  
Enteritis Virus

ABSTRACTMink enteritis virus (MEV), as a parvovirus, is among the smallest of the animal DNA viruses. The limited genome leads to multifunctional sequences and complex gene expression regulation. Here, we show that the expression of viral capsid protein 2 (VP2) of MEV requires its 5′ untranslated regions (5′ UTR) which promote VP2 gene expression at both transcriptional and translational levels. The expression of VP2 was inhibited in several common eukaryotic expression vectors. Our data showed that the 5′ UTR of VP2 enhanced capsid gene transcription but not increased stability or promotes nucleocytoplasmic export of VP2 mRNA. Analysis of the functions of 5′ UTR fragments showed that the proximal region (nucleotides [nt] 1 to 270; that is, positions +1 to +270 relative to the transcription initiation site, nt 2048 to 2317 of MEV-L) of 5′ UTR of VP2 was necessary for VP2 transcription and also promoted the activity of P38 promoter. Unexpectedly, further analysis showed that deletion of the distal region (nt 271 to 653) of the 5′ UTR of VP2 almost completely abolished VP2 translation in the presence of P38, whereas the transcription was still induced significantly. Furthermore, using a luciferase reporter bicistronic system, we identified that the 5′ UTR had an internal ribosome entry site-like function which could be enhanced by NS1 via the site at nt 382 to 447. Mutation of the 5′ UTR in the MEV full-length clones further showed that the 5′ UTR was required for VP2 gene expression. Together, our data reveal an undiscovered function of 5′ UTR of MEV VP2 in regulating viral gene expression.IMPORTANCEMEV, a parvovirus, causes acute enteritis in mink. In the present report, we describe an untranslated sequence-dependent mechanism by which MEV regulates capsid gene expression. Our results highlight the roles of untranslated sequences in regulating the transcriptional activity of P38 promoter and translation of capsid genes. These data also reveal the possibility of an unusual translation mechanism in capsid protein expression and the multiple functions of nonstructural protein. A better understanding of the gene expression regulation mechanism of this virus will help in the design of new vaccines and targets for antiviral agents against MEV.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

2009 ◽  
Vol 23 (4) ◽  
pp. 617-621 ◽  
Author(s):  
Martijn Vermeulen ◽  
Anne-Marie M.J.F. Boerboom ◽  
Barry M.G. Blankvoort ◽  
Jac M.M.J.G. Aarts ◽  
Ivonne M.C.M. Rietjens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
Glutathione S Transferase ◽  
Luciferase Reporter Gene ◽  
Responsive Element

scholarly journals Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1216 ◽  
Author(s):  
Juliette M. Delhove ◽  
Rajvinder Karda ◽  
Lorna M. FitzPatrick ◽  
Suzanne M.K. Buckley ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Viral Vectors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Whole Body ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Luciferase Reporter Gene ◽  
Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Quan He
Keyword(s):  
Gene Expression ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Phospholipase A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P450 Monooxygenase ◽  
Stimulation Of

P155 Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10 -7 M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA 2 , is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.

Interfering with Long Non-Coding RNA FBXL19-AS1 Inhibits the Proliferation, Invasion and Migration of Hepatoma Carcinoma Cells via Targeting miR-541-5p

2021 ◽  
Vol 11 (11) ◽  
pp. 2120-2127
Author(s):  
Weijun Lu ◽  
Qun Wang ◽  
Changbo Fu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Cell Lines ◽  
Reporter Gene ◽  
Malignant Tumors ◽  
Human Life ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and the morbidity and mortality of HCC rate in the first few malignant tumors, seriously threatening the safety of human life. LncRNA is a hot topic in tumor research in recent years. The abnormal expression of LncRNA FBXL19-AS1 and its potential target as a tumor diagnostic marker have been confirmed in colon cancer, breast cancer and lung cancer, etc. However, the study on LncRNA FBXL19-AS1 in HCC has not been reported. Rt-qPCR was used to detect the expression of FBXL19-AS1 and miR-541-5p in HCC cell lines, and luciferase reporter gene was used to detect whether there were binding sites between LncRNA FBXL19-AS1 and miR-541-5p. Interfered with FBXL19-AS1 and overexpressed miR-541-5p were detected by cell transfection. Then CCK-8 and colony formation assay were used to detect cell viability and cell proliferation. Wound healing detected the rate of cell migration and Transwell detected the rate of cell invasion. Western blot was used to detect the expression of proteins related to cell migration and invasion. The expression of FBXL19-AS1 in HCC cell lines was significantly higher than that in normal liver cells (LO2). Moreover, FBXL19-AS1 can promote HCC cell proliferation, migration and invasion. Luciferase reporter gene confirmed the binding site between LncRNA FBXL19-AS1 and miR-541-5p. After interfering with the expression of FBXL19-AS1, miR-541-5p was significantly increased. Subsequently, overexpression of miR-541-5p can inhibit the expression of lncRNA FBXL19-AS11 and promote proliferation, migration and invasion of hepatocellular carcinoma. So we can conclude that lncRNA FBXL19-AS1 promoted the proliferation, migration and invasion of HCC cells through targeting miR-541-5p.

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