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

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.

Screening of 19 traditional Mexican medicinal plants for effects on the metabolic syndrome related targets PPARα, β/δ, and γ in an in vitro luciferase reporter gene assay

Planta Medica ◽

10.1055/s-0035-1565656 ◽

2015 ◽

Vol 81 (16) ◽

Author(s):

K Kuchta ◽

N Matsuura ◽

HW Rauwald ◽

R Quintanilla-Licea ◽

M Iinuma

Keyword(s):

Metabolic Syndrome ◽

Medicinal Plants ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

The Metabolic Syndrome ◽

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

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/797496 ◽

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 ◽

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.

LINC01213 promotes prostate cancer cell progression through miR-597/ BCL2L1 axis

10.21203/rs.3.rs-289818/v1 ◽

2021 ◽

Author(s):

Xian Zhao ◽

Xiaojing Xu ◽

Qiong Wang ◽

Xiaofei Wu

Keyword(s):

Prostate Cancer ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Western Blot Assay ◽

Abstract Background: Majority of cancer related deaths in males are attributed to prostate cancer (PRAD) throughout the world. Recently, the role of long non-coding RNAs (lncRNAs) in the pathogenesis of cancer has been widely explored. In this study, we investigated the role of lncRNA LINC01213 (LINC01213) in tumorigenesis of prostate cancer (PRAD).Methods: PRAD and adjacent tissue samples were collected from cancer patients. Survival rate among these patients was compared by Kaplan–Meier analysis. PRAD cells viability was estimated by CCK-8 method while AnnexinV/PI cytometry assay was used to determine the percent of apoptotic cells. qRT-PCR and western blot assay were used to determine the mRNA and protein expressions, respectively. Interaction between LINC01213 and corresponding miRNA as well as between miRNA and mRNA was confirmed by dual luciferase reporter gene assay. PRAD cells were also injected subcutaneously in nude mice to support in vitro findings.Results: It was observed that LINC01213 was highly expressed in PRAD samples and cell lines. Down-regulation of LINC01213 in PRAD cells decreased cell viability and inhibited proliferation. Luciferase reporter gene assay and RNA pull-down confirmed that LINC01213 targeted miR-597-3p. Increased expression of miR-597-3p resulted in decreased BCL2L2 expression in vitro. Inhibitory effects of miR-597-3p on PRAD cells’ survival and growth were diminished after LINC01213 overexpression which was also associated with alteration in the protein expression of BCL-xL, BCL-2 as well as caspase 3 and caspase 9.Conclusion: Taken together, our findings suggest that LINC01213 plays its role in PRAD tumorigenesis through miR-597-3p/ BCL2L2 dependent pathway with associated modulation of genes involved in cell survival and apoptosis.

Effects of Leonurus japonicus Houtt. and its N-containing constituents leonurine and stachydrine on the activity of PPARα, β/δ, and γ in a newly developed in vitro luciferase reporter gene assay

Planta Medica ◽

10.1055/s-0034-1394682 ◽

2014 ◽

Vol 80 (16) ◽

Cited By ~ 1

Author(s):

K Kuchta ◽

N Matsuura ◽

HW Rauwald ◽

M Iinuma

Keyword(s):

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Μελέτη της επίδρασης της ελευρωπαΐνης στην ομοιοστασία των λιπιδίων

10.12681/eadd/44412 ◽

2017 ◽

Author(s):

Φωτεινή Μάλλιου

Keyword(s):

Reporter Gene ◽

In Silico ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Wild Type ◽

Η Ελευρωπαΐνη, το κύριο πολυφαινολικό συστατικό της ελιάς, παρουσιάζει αντιοξειδωτικές και υπολιπιδαιμικές ιδιότητες. Ο ενεργοποιημένος υποδοχέας επαγωγής του πολλαπλασιασμού των υπεροξεισωμάτων τύπου α (PPARα), διαδραματίζει καίριο ρόλο στον έλεγχο του μεταβολισμού των λιπιδίων και στην ενεργειακή ομοιαστασία του κυττάρου. Η συγκεκριμένη έρευνα εστιάζει στους μηχανισμούς της υπολιπιδαιμικής δράσης της Ελευρωπαΐνης με έμφαση στο ρόλο της Ελευρωπαΐνης στην ενεργοποίηση του PPARα. Για τον σκοπό αυτό, έγινε αξιολόγηση in silico της ικανότητας της Ελευρωπαΐνης να προσδένεται στον PPARα. Θεωρητικά μοντέλα πρόσδεσης στην κρυσταλλική δομή του PPARα με τη χρήση Μοριακής Προσομοίωσης Πρόσδεσης, επιβεβαιώνουν την υπόθεσή μας ότι η Ελευρωπαΐνη είναι αγωνιστής του PPARα. Επιπλέον, διερευνήθηκε in vitro με το Luciferase reporter gene assay η ικανότητα της Ελευρωπαΐνης να προσδένεται στον υποδοχέα PPARα και να τον ενεργοποιεί. Τα αποτελέσματα από την in silico και την in vitro μελέτη δείχνουν σαφώς ότι η ελευρωπαΐνη ενεργοποιεί τον PPARα. Στη συνέχεια έγινε in vivo διερεύνηση της ενεργοποίησης του PPARα από την Ελευρωπαΐνη και αξιολόγηση της επίδρασής της στο λιπιδαιμικό προφίλ των πειραματόζωων. Αγωγή αρσενικών μυών άγριου τύπου (SV129 Wild Type) με Ελευρωπαΐνη σε δοσολογία 100mg/kg, p.o, τα οποία ακολούθησαν τυπική δίαιτα για τρωκτικά, για 6 εβδομάδες, είχε ως αποτέλεσμα επαγωγή του Pparα και των γονιδίων-στόχων του στο ήπαρ, πιθανώς μέσω ενεργοποίησης του PI3K/AKT/p70S6K σηματοδοτικού μονοπατιού. Αυτή η επίδραση της Ελευρωπαΐνης φαίνεται να σχετίζεται άμεσα με σημαντική μείωση των επιπέδων των TGs του ορού και της ολικής χοληστερόλης, γιατί η Ελευρωπαΐνη δεν είχε καμία επίδραση σε αυτούς τους λιπιδαιμικούς δείκτες σε διαγονιδιακούς Pparα null μύες. Στην κατεύθυνση της διερεύνησης της επίδρασης της Ελευρωπαΐνης σε ηπατικούς παράγοντες και σε παράγοντες, που εκφράζονται στο λευκό λιπώδη, κρίσιμους για την ομοιοστασία των Τριγλυκεριδίων, διαπιστώθηκαν τα ακόλουθα: 1) Ενεργοποίηση της ορμονο-ευαίσθητης λιπάσης (HSL) στο λευκό λιπώδη ιστό (W.A.T.) των άγριου τύπου μυών, 2) επαγωγή ποικίλλων ηπατικών παραγόντων, που συμμετέχουν στη σύνθεση, τη μεταφορά, τον μεταβολισμό και την απέκκριση των τριγλυκεριδίων. Αυτή η ενεργοποίηση της HSL στον W.A.T. και των ηπατικών παραγόντων, που συμμετέχουν στην ομοιοστασία των λιπιδίων, είναι πιθανόν να ενισχύει την επαγόμενη από την Ελευρωπαΐνη μείωση των επιπέδων των τριγλυκεριδίων και της χοληστερόλης στον ορό. Συνοψίζοντας, φαίνεται ότι η Ελευρωπαΐνη μειώνει τα τριγλυκερίδια και την ολική χοληστερόλη του ορού σε μύες μέσω ενεργοποίησης του PPARα. Τα δεδομένα αυτής της μελέτης δείχνουν επίσης, ότι στην υπολιπιδαιμική δράση της Ελευρωπαΐνης συμμετέχει και η ενεργοποίηση της HSL στο λευκό λιπώδη ιστό καθώς και η επαγωγή ηπατικών γονιδίων, που διαδραματίζουν βασικό ρόλο την ομοιοστασία των τριγλυκεριδίων, δηλαδή τη σύνθεση, την μεταφορά, τον μεταβολισμό και την κάθαρσή τους. Συμπερασματικά, η μελέτη έδειξε τις υπολιπιδαιμικές δυνατότητες της Ελευρωπαΐνης σε μύες και αποσαφήνισε σε σημαντικό βαθμό τους μηχανισμούς της υπολιπιδαιμικής δράσης της, φωτίζοντας κυρίως τον ρόλο του PPARα. Επειδή η διατήρηση της ομοιοστασίας των λιπιδίων είναι μια πολύπλοκη διαδικασία, που ρυθμίζεται από πολλούς παράγοντες, πιστεύουμε ότι πιθανώς εμπλέκονται και άλλοι μηχανισμοί στην δράση της Ελευρωπαΐνης, η διερεύνηση των οποίων είναι αντικείμενο μελλοντικών μελετών.

Effects of medicinal plants of the genus Leonurus and seventeen of their isolated constituents on the activity of PPARα, β/δ, and γ in an in vitro luciferase reporter gene assay

Planta Medica ◽

10.1055/s-0035-1565632 ◽

2015 ◽

Vol 81 (16) ◽

Author(s):

K Kuchta ◽

N Matsuura ◽

HW Rauwald ◽

C Rusch ◽

Y Shoyama ◽

...

Keyword(s):

Medicinal Plants ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.2314 ◽

2011 ◽

Vol 2 (1) ◽

pp. 11

Author(s):

Darja Henseler ◽

Jonathan D. Turner ◽

Matthias Eckhardt ◽

Maaike Van der Mark ◽

Yanina Revsin ◽

...

Keyword(s):

Reporter Gene ◽

In Silico ◽

Promoter Activity ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Regulatory Structure ◽

Promoter Regions ◽

Potential Promoter

The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213103513 ◽

2020 ◽

Vol 20 (6) ◽

pp. 715-723

Author(s):

Natarajan Nandakumar ◽

Pushparathinam Gopinath ◽

Jacob Gopas ◽

Kannoth M. Muraleedharan

Keyword(s):

Synergistic Effect ◽

Hodgkin’S Lymphoma ◽

A549 Cells ◽

Hodgkin's Lymphoma ◽

Luciferase Reporter ◽

Lymphoma Cells ◽

Nuclear Extracts ◽

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽

10.3233/tub-200072 ◽

2021 ◽

Vol 43 (1) ◽

pp. 11-26

Author(s):

Maike Busch ◽

Natalia Miroschnikov ◽

Jaroslaw Thomas Dankert ◽

Marc Wiesehöfer ◽

Klaus Metz ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treated Patient ◽

Luciferase Reporter ◽

Gene Assay ◽

The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

MiR-206 regulates the progression of osteoporosis via targeting HDAC4

European Journal of Medical Research ◽

10.1186/s40001-021-00480-3 ◽

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 ◽

Mineral Density ◽

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.