scholarly journals Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Qun Chen ◽  
Hai-tang Xie ◽  
Yan Li ◽  
Guo Wang ◽  
Zhe Xu ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Reporter Gene ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Expression Plasmid

Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites.Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit.Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P>0.05).Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.

scholarly journals miR-16-5p Regulates PTPN4 and Affects Cardiomyocyte Apoptosis and Autophagy Induced by Hypoxia/Reoxygenation

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zheng Cao ◽  
Jinglan Liu ◽  
Zhanqing Zhao ◽  
Qiao Wang
Keyword(s):  
Reporter Gene ◽  
Cell Apoptosis ◽  
Cell Damage ◽  
Negative Control Group ◽  
Negative Control ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Hypoxia Reoxygenation

Objectives. To explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment. Methods. AC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4. Results. After H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly ( P < 0.05 ), and the levels of cell apoptosis and autophagy also increased significantly ( P < 0.05 ). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly ( P < 0.05 ). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced ( P < 0.05 ), and the rates of cell apoptosis and autophagy were also significantly reduced ( P < 0.05 ). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells ( P < 0.05 ), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment ( P < 0.05 ), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells ( P < 0.05 ). Conclusions. The expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

scholarly journals A Novel Mechanism of bta-miR-210 in Bovine Early Intramuscular Adipogenesis

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 601
Author(s):  
Ling Ren ◽  
Qian Li ◽  
Xin Hu ◽  
Qiyuan Yang ◽  
Min Du ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Adipogenic Differentiation ◽  
Growth Factor Receptor ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Intracellular Lipid Accumulation ◽  
Major Factors

Intramuscular fat (IMF) is one of the major factors determining beef quality. IMF formation is influenced by multiple conditions including genetic background, age and nutrition. In our previous investigation, bta-miR-210 was found to be increased during adipogenesis using miRNA-seq. In this study, we validated the upregulation of bta-miR-210 in platelet-derived growth factor receptor α positive (PDGFRα+) progenitor cells during adipogenic differentiation in vitro. To investigate its role in adipogenesis, bta-miR-210 mimics were introduced into progenitor cells, which resulted in enhanced intracellular lipid accumulation. Accordingly, the expression of adipocyte-specific genes significantly increased in the bta-miR-210 mimic group compared to that in the negative control group (p < 0.01). Dual-luciferase reporter assays revealed that WISP2 is a target of bta-miR-210. WISP2 knockdown enhanced adipogenesis. In conclusion, bta-miR-210 positively regulates the adipogenesis of PDGFRα+ cells derived from bovine fetal muscle by targeting WISP2.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals In vitro comparison of the cytotoxicity of different endodontic sealers; AH Plus, AdSeal, Endoseal MTA, and GuttaFlow Bioseal

2021 ◽  
Author(s):  
Mehdi Dastorani ◽  
Muhammad javad Aliee ◽  
Raheleh Halabian ◽  
Mostafa Solati ◽  
Mohammadsadegh Alemrajabi
Keyword(s):  
Repeated Measures ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Gingival Fibroblasts ◽  
Periapical Lesions ◽  
Ah Plus ◽  
Low Cytotoxicity ◽  
Endodontic Sealers

Abstract Background: This study aimed to assess the cytotoxicity of four commonly used endodontic sealers namely AH Plus, AdSeal, Endoseal MTA, and GuttaFlow Bioseal against human gingival fibroblasts (HGFs). Methods: After culturing the HGFs, they were exposed to the respective sealers in set form and in five different weights, after sterilization. The cytotoxicity of the sealers was evaluated after 1, 3 and 7 days using the methyl thiazolyl tetrazolium (MTT) assay. Data were analyzed by repeated measures ANOVA. Results: After 24 h, all sealers showed low cytotoxicity. However, all sealers in 250 mg and 500 mg weights showed significantly higher cytotoxicity than the negative control group at 72 h, and 7 days (P<0.05) except for AdSeal in 80 mg weight (P>0.05). AH Plus was significantly more cytotoxic than other sealers at 3 and 7 days (P<0.05) while AdSeal had the closest results to the negative control group, and showed significantly higher biocompatibility than other sealers in 250 mg concentration. Conclusion: AdSeal showed the highest biocompatibility while AH Plus had the highest cytotoxicity among the tested sealers. Thus, its application may delay the healing of periapical lesions.

scholarly journals Overexpression of miRNA-21 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma Cells via Suppressing SMAD7

2019 ◽  
Vol 18 ◽  
pp. 153303381987868 ◽  
Author(s):  
Yan Wang ◽  
Ping Zhang ◽  
Mei Yuan ◽  
Xiaojie Li
Keyword(s):  
Hepatocellular Carcinoma ◽  
Family Member ◽  
Reporter Gene ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Hepatoma Cell ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Proliferation And Invasion

Purpose: This study aimed to explore the molecular mechanism of microRNA-21 and smad family member 7 in hepatocellular carcinoma. Method: A total of 57 participants were divided into control group (healthy participants, n = 10) and hepatocellular carcinoma group (hepatocellular carcinoma patients, n = 37). The expression of microRNA-21 levels were first detected in these two groups. Cell transfection was performed on hepatoma cell lines, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay to reveal proliferation and invasion ability. Furthermore, the relation between microRNA-21 and smad family member 7 was revealed by luciferase reporter gene and RNA immunoprecipitation assay. Finally, a transplantation tumor model of breast cancer in mice was constructed. Results: The serum indicators including α-alanine aminotransferase, aspartate aminotransferase, and albumin were differentially expressed between hepatocellular carcinoma group and control group. Compared to the control group, there was a high expression of microRNA-21 in hepatocellular carcinoma group. Low expression of microRNA-21 inhibited the proliferation and invasion of HepG2.2.15 and Huh7-1.3 cells. Luciferase reporter gene and RNA innumoprecipitation assay showed that smad family member 7 was the target gene of microRNA-21. Moreover, mice model analysis showed that microRNA-21 might regulate the growth of the transplanted tumors in mice by targeting smad family member 7. Conclusion: The upregulated microRNA-21 might participate in the proliferation and migration in cells of hepatocellular carcinoma via suppression of smad family member 7. Furthermore, serum indicators such as alanine aminotransferase, aspartate aminotransferase, and albumin might be used as serum diagnostic markers for hepatocellular carcinoma.

scholarly journals Effect of different periodontal ligament simulating materials on the incidence of dentinal cracks during root canal preparation

2018 ◽  
Vol 12 (3) ◽  
pp. 196-200
Author(s):  
Ajita Rathi ◽  
Prateeksha Chowdhry ◽  
Mamta Kaushik ◽  
Pallavi Reddy ◽  
Roshni Roshni ◽  
...  
Keyword(s):  
Root Canal ◽  
Periodontal Ligament ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
P Value ◽  
Root Canal Preparation ◽  
Impression Material ◽  
Group 1

Background. The present study was undertaken to evaluate the incidence of dentinal cracks during root canal preparation with different periodontal ligament simulating materials in vitro. Methods. Seventy freshly extracted human mandibular first premolars were selected and divided into 7 groups in terms of simulating material: group 1: polyether impression material; group 2: polyvinyl acetate adhesive; group 3: polyvinyl siloxane impression material; group 4: cyanoacrylate adhesive; group 5: epoxy resin adhesive; group 6: positive control, without any periodontal ligament simulation; and group 7: negative control, where neither a periodontal ligament simulating material was used nor canal preparation was carried out. Root canal preparation was carried out in all the groups followed by sectioning of roots at 3 mm, 6 mm and 9 mm. The sections were evaluated under a stereomicroscope at ×2.5 for the presence or absence of cracks. Chi-squared test was used to compare the appearance of defective roots between the different experimental groups. Results. The least number of cracks were found in the negative control group, followed by group 1 where polyether impression material was used for periodontal ligament simulation. The difference was significant with a P-value of 0.002 for coronal sections. Conclusion. Under the limitation of the present study, polyether and polyvinyl siloxane (light body) can both be used for simulation of periodontal ligament.

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.

Cytotoxicity and Hemolytic Properties of Nano-Hydroxyapatite/Polyetheretherketone Biocomposites

2016 ◽  
Vol 848 ◽  
pp. 567-572 ◽  
Author(s):  
Lin Wang ◽  
Shen Hua Song ◽  
Zheng Zhi Wu ◽  
Li Hong Duan ◽  
Chun Bao Wang
Keyword(s):  
Negative Control Group ◽  
Negative Control ◽  
National Standard ◽  
Control Group ◽  
Culture Solution ◽  
Contact Method ◽  
Cytotoxicity Test ◽  
L929 Cells ◽  
Hemolysis Test

In order to evaluate the cytocompatibility and hemolytic properties of n-HA/PEEK biocomposites the nanohydroxyapatite/polyetheretherketone (n-HA/PEEK) biocomposites were successfully prepared. The mechanical properties of the biocomposites were proximal to human bone, at the same time, they had the optimal value with the HA volume content of 5%. The PEEK and n-HA/PEEK biocomposites with different HA content extraction medium was prepared with fresh medium. Simple DMEM culture solution was taken as negative control group. The pure PEEK and 5vol.%, 15vol.%, 30vol.% n-HA/PEEK biocomposites were the testing group. The relative proliferation rate of L929 cells was determined on the 1st, 2nd, 3rd and 6th days with CCK-8 assay. The cytotoxicity of n-HA/PEEK biocomposites were evaluated according to ISO 10993-5: 2009. The L929 cells morphology and growth on the 1st, 2nd, 3rd and 6th days were determined under inverted microscope. The hemolysis test in vitro of n-HA/PEEK biocomposites were evaluated through measuring erythrocyte lysis and ferro-hemoglobin freeing degree with indirect contact method basing on ISO 10993-4:2009. The experimental results showed that the growth and morphology of cells in pure PEEK and n-HA/PEEK biocomposites extraction medium had no difference from negative control group. Cytotoxicity test showed that PEEK and n-HA/PEEK biocomposites did not have obvious toxicity on L929 cells, and the cytotoxicity of these extracts was in grade 0-1. Hemolysis test suggested that PEEK and n-HA/PEEK biocomposites did not have obvious hemolysis reaction, and the hemolysis rate of PEEK and n-HA/PEEK biocomposites were 2.37%, 1.71%, 1.05% and 1.32% respectively, which are less than the national standard (5%). It may be concluded that the n-HA/PEEK biocomposites did not have obvious cytotoxicity and hemolysis reaction, which demonstrated that n-HA/PEEK biocomposites had good cytocompatibility.

scholarly journals Potential of Cocoa Extract (Theobroma cacao) in Inhibiting Erythrocyte Damage Induced by Physalia utriculus Venom

2020 ◽  
Vol 6 (3) ◽  
pp. 157
Author(s):  
Adinningtyas Intansari ◽  
Al Munawir ◽  
Laksmi Indreswari
Keyword(s):  
Theobroma Cacao ◽  
Ethanol Extract ◽  
Negative Control Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Marine Biota ◽  
Erythrocyte Lysis ◽  
Theobroma Cacao L

Physalia utriculus is one of the invertebrate marine biota that is often found in Indonesia. Some symptoms of venoming due to jellyfish stings cause pain, itching, and hemolysis. In Indonesia, 13 cases of jellyfish stings were reported in 2005-2009 with three people dying in Java, Bali, and Bangka. Cocoa beans (Theobroma cacao L.) contain fat, carbohydrates, proteins, and polyphenol compounds that are useful as antioxidants. Polyphenols in the form of epicathechins, catechins, and procyanidins serve to provide protection to hemolysis. The purpose of this study was to determine the potential of ethanol extract of cacao (Theobroma cacao L.) in inhibiting the damage of erythrocyte induced by Physalia utriculus in vitro. This study used 28 samples of erythrocytes divided into seven groups, namely the normal control group, negative controls, and treatment with cocoa ethanol extract 0.2%, 0.1%, 0.04%, and 0.02%. Each subsequent group induced venom Physalia utriculus. The results showed that the average speed of erythrocyte lysis in the treatment group by giving cocoa ethanol extract 0.2%, 0.1%, 0.04%, and 0.02% respectively (seconds ± standard deviation) was 858,25 ± 94,44; 1.000,5 ± 159,93; 678,5 ± 19,71; and 1.006 ± 159,50. The mean speed of erythrocyte lysis in the negative control group was 1,025 ± 164.63 and the positive control group with the administration of N-Acetylcystein can last up to one hour after administration of venoms. Test for normality and homogeneity shows that data is normally distributed and homogeneous. One Way Annova analysis shows the significance value of p <0.05, then a post hoc analysis test was performed with the Bonferoni method to find out the differences in significance in each group. In this study it can be concluded that the administration of cocoa ethanol extract has no potential to inhibit erythrocyte damage that has been venomed by Physalia utriculus in vitro. Keywords: Physalia utriculus, cacao, erythrocyte damage

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