scholarly journals Quantitative Assessment of Pdx1 Promoter Activity in Vivo Using a Secreted Luciferase Reporter System

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 4388-4395 ◽  
Author(s):  
Wataru Nishimura ◽  
Koki Eto ◽  
Atsushi Miki ◽  
Motohito Goto ◽  
Miho Kawaguchi ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Quantitative Assessment ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System

scholarly journals MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Malignant Tumors ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most common and malignant tumors in the brain of humans, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by regulating gene expression at the posttranscriptional levels. Although miR-218 has been reported to be downregulated in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression in gliomas and normal brain tissues (control subjects) were analyzed using TCGA dataset. The biological roles of miR-218 in glioma cells were determined by a series of in vitro and in vivo studies. The dual-luciferase reporter system was performed to identify potential targets of miR-218. The regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway was evaluated by dual-luciferase reporter system and western blot.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and exerted a potent tumor suppressor in glioma cells by inhibiting cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 in glioma by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop.

scholarly journals MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Regulation Of Gene Expression ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most malignant and common tumors in human brains, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by performing posttranscriptional regulation of gene expression. Although miR-218 has been demonstrated decreased in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression were analyzed in gliomas and normal brain tissues (control subjects) using TCGA dataset. A series of in vitro and in vivo studies was performed to determine the biological roles of miR-218 in glioma cells. Potential targets of miR-218 were identified using dual-luciferase reporter system. Western blot and dual-luciferase reporter system were performed to evaluate the regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and played potent tumor suppressor roles in glioma cells by inhibited cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop in glioma.

scholarly journals miR-30a as Potential Therapeutic by Targeting TET1 through Regulation of the Hydroxymethlation of Drp-1 Promoter in IPF

2017 ◽  
Author(s):  
Songzi Zhang ◽  
Huizhu Liu ◽  
Yuxia Liu ◽  
Jie Zhang ◽  
Hongbo Li ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Target Gene ◽  
Mitochondrial Fission ◽  
Luciferase Reporter ◽  
Biological Processes ◽  
Reporter System ◽  
Potential Therapeutic Target ◽  
Direct Target ◽  
Luciferase Reporter System

Several recent studies have indicated that miR-30a plays critical roles in various biological processes and diseases. However, the mechanism of miR-30a participation in the regulation of idiopathic pulmonary fibrosis (IPF) is ambiguous. Our previous study demonstrated that miR-30a may function as a novel therapeutic target for lung fibrosis by blocking mitochondrial fission, which is dependent on dynamin-related protein-1 (Drp-1). However, the regulatory mechanism between miR-30a and Drp-1 has yet to be investigated. In addition, whether miR-30a can act as a potential therapeutic has not been verified in vivo. In this study, the miR-30a expression in IPF patients was evaluated. Computational analysis and a dual luciferase reporter system assay were used to identify the target gene of miR-30a, and cell transfection was used to confirm this relationship. Ten-eleven translocation 1 (TET1) was validated as a direct target of miR-30a, and the transfection of miR-30a mimic/inhibitor significantly reduced/increased the expression of TET1 protein. Further experiment verified that the interference on TET1(siRNA) could inhibit the hydroxymethlation of the Drp-1 promoter. Finally, miR-30a agomir was designed and applied to identify and validate the therapeutic effect of miR-30a in vivo. Our study demonstrated that miR-30a could inhibit the TET1 expression by base pairing with complementary sites in the 3′ untranslated region to regulate the hydroxymethlation of the Drp-1 promoter. Furthermore, miR-30a could act as a potential therapeutic target for IPF.

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

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