ABSTRACT
OBJECTIVE@#To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects.@*METHODS@#Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice.@*RESULTS@#Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P < 0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P < 0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P < 0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P < 0.05), and co-transfection with LV-PTEN obviously reversed these changes (P < 0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P < 0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P < 0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis.@*CONCLUSION@#Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.
Subject(s)
Humans , Animals , Mice , MicroRNAs/metabolism , Cell Line, Tumor , Cell Proliferation/physiology , Mice, Nude , Cell Movement/physiology , Colorectal Neoplasms/pathology , Luciferases/metabolism , Gene Expression Regulation, Neoplastic , PTEN Phosphohydrolase/metabolism , Chemokine CXCL12/metabolismABSTRACT
BACKGROUND: Previous studies have shown that long noncoding RNA (IncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this IncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR- 490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.
Subject(s)
Humans , Animals , Male , Stomach Neoplasms/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , Stomach Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Cell Movement , Cell Survival , Apoptosis , Xenograft Model Antitumor Assays , MicroRNAs/genetics , Cell Line, Tumor/metabolism , Epithelial Cells/metabolism , RNA, Long Noncoding/genetics , Carcinogenesis/metabolism , Luciferases/metabolism , Mice, Inbred BALB CABSTRACT
BACKGROUND: Breast cancer is the second common malignant cancer among females worldwide. Accumulating studies have indicated that deregulation of miRNA expression in breast cancer will contribute to tumorigenesis and form different cancer subtypes. However, the reported studies on miR-29b-3p-regulated breast cancer are limited so far. Herein, we investigated the role and mechanism of miR-29b-3p in the triple negative breast cancer cell line MDA-MB-231. METHODS: The relative miR-29b-3p expression in different breast cancer cell lines were determined by qRT-PCR. CCK8 and colony formation assay were used to determine the influence of miR-29b-3p on cell proliferation. Migration assay and invasion assay were performed for cell migration and invasion respectively. To study the cell integrity immunofluorescence was performed. TUNEL assay, flow cytometry assay, hoechst staining and western blot were conducted to determine the influence of miR-29b-3p inhibitor on cell apoptosis. TRAF3 was found to be the target gene of miR-29b-3p using bioinformatics predictions. Dual-luciferase assay was performed to determine the relative luciferase activity in NC, miR-29b-3p mimic, miR-29b-3p inhibitor with TRAF3 3'-UTR wt or TRAF3 3'-UTR mt reporter plasmids. The proteins expression of NF-κB signaling pathway in MDA-MB-231 after transfection with NC, miR-29b-3p mimic, miR-29b-3p inhibitor were determined by western blot. RESULTS: The miR-29b-3p expression was significantly increased in MDA-MB-231 compare with MCF-10A. miR-29b-3p inhibitor reduced the cell viability of MDA-MB-231 and inhibited cell migration and invasion. Cell cytoskeleton integrity destroyed after miR-29b-3p inhibitor treatment. Furthermore, we identified the mechanism and found miR-29b-3p targets the TRAF3 and activates NF-κB signaling pathway. CONCLUSIONS: From the above studies, our results indicated that miR-29b-3p acts as a promoter for the development of MDA-MB-231.
Subject(s)
Humans , Female , Down-Regulation/genetics , Apoptosis/drug effects , MicroRNAs/metabolism , TNF Receptor-Associated Factor 3/metabolism , Triple Negative Breast Neoplasms/genetics , Blotting, Western , Cell Line, Tumor , TNF Receptor-Associated Factor 3/genetics , Cell Proliferation , Triple Negative Breast Neoplasms/pathology , Luciferases/metabolismABSTRACT
ABSTRACT Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.
Subject(s)
Animals , Male , Female , Coleoptera/physiology , Coleoptera/chemistry , Luminescence , Luciferases/metabolism , Behavior, Animal , Brazil , Forests , Evolution, Molecular , Luciferases/chemistryABSTRACT
The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).
Subject(s)
Humans , Gene Expression Regulation, Neoplastic/genetics , Cyclooxygenase 2/genetics , Haplotypes , Up-Regulation , Mutagenesis, Site-Directed , Polymorphism, Single Nucleotide , Cell Line, Tumor , Cyclooxygenase 2/metabolism , MCF-7 Cells , Genotype , Luciferases/metabolismABSTRACT
OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .
OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .
Subject(s)
Animals , Humans , Early Growth Response Protein 1/genetics , Epithelial Cells/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , /metabolism , Sulindac/analogs & derivatives , Apoptosis/drug effects , Blotting, Western , Butadienes/pharmacology , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Early Growth Response Protein 1/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Imidazoles/pharmacology , Intestines/cytology , Intestines/drug effects , Intestines/metabolism , Luciferases/genetics , Luciferases/metabolism , Microscopy, Confocal , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , /antagonists & inhibitors , Nitriles/pharmacology , Pyridines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Sulindac/pharmacology , Transfection , Up-Regulation/drug effects , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolismABSTRACT
O objetivo deste artigo é investigar relações entre renda e escolaridade com condições de saúde e nutrição em obesos graves. Estudo transversal ambulatorial com 79 pacientes de primeira consulta, com Índice de Massa Corporal (IMC) ≥ 35 kg/m2 e idade ≥ 20 anos. Coletaram-se dados: sociodemográficos, antropométricos, estilo de vida, exames bioquímicos e consumo alimentar. O IMC médio foi 48,3 ± 6,9 kg/m2. Observou-se correlação negativa significante de escolaridade com variáveis peso (r = -0,234) e IMC (r = -0,364) e de renda familiar per capita com consumo diário de vegetal A (r = -0,263). Após análise multivariada maior renda familiar per capita se associou à ausência de cardiopatia (RP: 0,51, IC95%: 0,32-0,81), maior consumo diário de vegetal A (RP: 1,79, IC95%: 1,16-2,75) e doces (RP: 3,12, IC95%: 1,21-8,04). Em obesos graves a maior renda familiar per capita se associou à ausência de cardiopatia e maior consumo de vegetais folhosos e doces. Já a escolaridade não se manteve associada às condições de saúde e nutrição.
This article seeks to investigate the relationship between income and educational level and health and nutritional conditions among the morbidly obese. A cross-sectional study was conducted with 79 patients at first appointment, with Body Mass Index (BMI) ≥ 35 kg/m2 and age ≥ 20 years. The following data was collected: demographic, socioeconomic, anthropometric, lifestyle, biochemical and food intake data. Average BMI was 48.3 ± 6.9 kg/m2. There was a significant negative correlation between education level and the variables of weight (r = -0.234) and BMI (r = -0.364) and per capita family income with daily consumption of leafy vegetables (r = -0.263). After multivariate analysis, higher per capita family income was associated with the absence of heart disease (PR: 0.51, CI95%: 0.32-0.81), higher daily consumption of leafy vegetables (PR: 1.79, CI95%: 1.16-2.75) and candy (PR: 3.12, CI95%: 1.21-8.04). In the morbidly obese, per capita household income was associated with absence of heart disease and higher consumption of leafy vegetables and candy. On the other hand, education level was not associated with health and nutrition conditions.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Phospholipases A/metabolism , /pharmacology , /pharmacology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Glucuronidase/metabolism , Luciferases/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phospholipases A/antagonists & inhibitors , Protein Processing, Post-Translational/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seedlings/drug effects , Seedlings/metabolism , Time FactorsABSTRACT
BACKGROUND: Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant. RESULTS: In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes. CONCLUSION: Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.
Subject(s)
Humans , Brain Neoplasms/pathology , Membrane Glycoproteins/pharmacology , Apoptosis , Semaphorins/pharmacology , MicroRNAs/antagonists & inhibitors , Cell Proliferation , Glioma/pathology , Brain Neoplasms/metabolism , Membrane Glycoproteins/genetics , Signal Transduction , Gene Expression Regulation, Neoplastic , Cell Movement , Blotting, Western , Semaphorins/genetics , MicroRNAs/metabolism , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , Glioma/metabolism , Luciferases/metabolism , Neoplasm InvasivenessABSTRACT
PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.
Subject(s)
Animals , Male , Mice , Diabetes Mellitus, Experimental/complications , Electroporation/methods , Erectile Dysfunction/therapy , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Genetic Therapy/methods , Luciferases/metabolism , Mice, Inbred C57BL , Penile Erection/physiology , Penis/physiopathology , TransfectionABSTRACT
PURPOSE: The aim of the present study was to investigate associations between the renin gene (REN) and the risk of essential hypertension and blood pressure (BP) levels in Koreans. MATERIALS AND METHODS: To outline the functional role of a single nucleotide polymorphism in the transcription of the REN gene, we conducted a case-control study of 1975 individuals: 646 hypertension (HT) patients and 1329 ethnically and age-matched normotensive subjects. RESULTS: Logistic regression analysis indicated that the genotypes AA/AG were strongly associated with risk of HT (odds ratio, 1.493; 95% confidence interval, 1.069-2.086, p=0.018) in female subjects. The genotypes AA/AG also showed significant association with higher blood pressure levels, both systolic and diastolic, in postmenopausal HT women (p=0.003 and p=0.017, respectively). Analysis of the promoter containing rs6682082 revealed a 2.4+/-0.01-fold higher activity in the A variant promoter than the G variant promoter, suggesting that rs6682082 is itself a functional variant. CONCLUSION: We suggest that the A allele of rs6682082 is a positive genetic marker for predisposition to essential hypertension and high BP in Korean women and may be mediated through the transcriptional activation of REN.
Subject(s)
Female , Humans , Middle Aged , Alleles , Asian People/genetics , Blood Pressure/genetics , Case-Control Studies , Diastole/genetics , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Hypertension/genetics , Luciferases/metabolism , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Renin/genetics , Republic of Korea , Risk Factors , Systole/genetics , TransfectionABSTRACT
Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 x 10(-7)). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 x 10(-5)). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 +/- 10(-10)). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.
Subject(s)
Female , Humans , Male , Middle Aged , Apolipoproteins/genetics , Arthritis, Rheumatoid/genetics , Case-Control Studies , DNA/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study , Genotype , Heterozygote , Homozygote , Lipocalins/genetics , Luciferases/metabolism , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Risk FactorsABSTRACT
Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64 percent, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.
Subject(s)
Humans , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Indoles/antagonists & inhibitors , Oximes/antagonists & inhibitors , Signal Transduction/drug effects , Wnt Proteins/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Cell Proliferation/drug effects , Drug Evaluation, Preclinical/methods , Genes, Reporter/physiology , Jurkat Cells , Luciferases/metabolism , Plasmids/drug effects , Plasmids/genetics , Transfection/methods , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/analysis , Animals , Coleoptera , Biochemistry/methods , Brain/metabolism , Female , Firefly Luciferin/metabolism , Luciferases/metabolism , Luminescent Measurements , Mice , Mice, Inbred C57BL , Sensitivity and Specificity , Time FactorsABSTRACT
Diseases activate the innate immune response which causes ancillary damage to the human body. Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g. DNA, RNA, protein, lipids and sugars. The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage. In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation. (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants. (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants. We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation. In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.
Subject(s)
Antioxidants/analysis , Luminescent Measurements , DNA Damage , DNA, Superhelical , Escherichia coli/genetics , Fruit/chemistry , Luciferases/metabolism , Luminol/chemistry , Molsidomine/analogs & derivatives , Mutation , Nitrates/analysis , Oxidants/chemistry , Oxidation-Reduction , Peroxynitrous Acid/chemical synthesis , Phenols/chemistry , Plasmids , Solutions , Tea/chemistryABSTRACT
In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.
Subject(s)
Animals , Female , Mice , Comparative Study , DNA/administration & dosage , Drug Delivery Systems , Electric Conductivity , Electroporation/methods , Escherichia coli/genetics , Genetic Therapy/methods , Gene Transfer Techniques , Genes, Reporter , Injections, Intramuscular , Luciferases/metabolism , Mice, Inbred BALB C , Muscle, Skeletal/drug effects , Osmolar Concentration , Plasmids/genetics , Sodium Chloride/pharmacology , Transfection , Pharmaceutical Vehicles/administration & dosage , beta-Galactosidase/metabolismABSTRACT
In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression in-creased as cation concentration of vehicle dec-reased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.
Subject(s)
Animals , Female , Mice , Comparative Study , DNA/administration & dosage , Drug Delivery Systems , Electric Conductivity , Electroporation/methods , Escherichia coli/genetics , Genetic Therapy/methods , Gene Transfer Techniques , Genes, Reporter , Injections, Intramuscular , Luciferases/metabolism , Mice, Inbred BALB C , Muscle, Skeletal/drug effects , Osmolar Concentration , Plasmids/genetics , Sodium Chloride/pharmacology , Transfection , Pharmaceutical Vehicles/administration & dosage , beta-Galactosidase/metabolismABSTRACT
Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression
Subject(s)
Humans , Adult , Fetal Hemoglobin/genetics , Genes, Reporter , Globins/genetics , Hemoglobinopathies/genetics , In Vitro Techniques , Mutation , beta-Galactosidase/metabolism , DNA Primers , Gene Expression , Globins/metabolism , Luciferases/genetics , Luciferases/metabolism , Point Mutation , Polymerase Chain Reaction , TransfectionABSTRACT
A possible role of hepatocyte nuclear factor 1 (HNF1) or HNF3, a predominant trans-acting factors of hepatic or pancreatic beta-cells, was examined on the tissue specific interdependent expression of glucokinase (GK) in liver, H4IIE, HepG2, HIT-T15 and MIN6 cell line. The tissues or cell lines known to express GK showed abundant levels of HNF1 and HNF3 mRNA as observed in liver, H4IIE, HepG2, HIT-T15 and MIN6 cells, whereas they were not detected in brain, heart, NIH 3T3, HeLa cells. The promoter of glucokinase contains several HNF3 consensus sequences and are well conserved in human, mouse and rat. Transfection of the glucokinase promotor linked with luciferase reporter to liver or pancreatic beta cell lines showed high interacting activities with HNF1 and HNF3, whereas minimal activities were detected in the cells expressing very low levels of HNFs. The binding of HNF1 or HNF3 to the GK promoter genes was confirmed by electrophoretic mobility shift assay (EMSA). From these data, we propose that the expression of HNF1 and/or HNF3 may, in part, contribute to the tissue specific expression of GK.
Subject(s)
Humans , Mice , Rats , 3T3 Cells , Animals , Blotting, Northern , Cell Line , Cell Nucleus/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , Genes, Reporter , Glucokinase/biosynthesis , HeLa Cells , Liver/metabolism , Luciferases/metabolism , Models, Genetic , Nuclear Proteins/genetics , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Tissue Distribution , Transcription Factors/genetics , Transcription, Genetic , TransfectionABSTRACT
We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.