scholarly journals Diminished YAP1 affects mitochondrial dynamics in IDH1 mutant glioma

2021 ◽  
Author(s):  
Shruti Patrick ◽  
Pruthvi Gowda ◽  
Kirti Lathoria ◽  
Vaishali Suri ◽  
Ellora Sen
Keyword(s):  
Mitochondrial Dynamics ◽  
Redox Homeostasis ◽  
Luciferase Reporter ◽  
Ros Generation ◽  
Mitochondrial Fragmentation ◽  
Isocitrate Dehydrogenase 1 ◽  
Idh1 R132h ◽  
Important Regulator ◽  
Reporter Assays ◽  
Induced Apoptosis

Mutation in isocitrate dehydrogenase 1 (IDH1) gene, leading to the production of oncometabolite D-2-hydroxyglutarate (2-HG) from α-ketoglutarate, is associated with better prognosis in glioma. As Yes-associated protein 1 (YAP1) is an important regulator of tumor progression, its role in glioma expressing IDH1 R132H mutation was investigated. Diminished nuclear YAP1 in IDH1 mutant patient gliomas and cell lines was accompanied by decreased TFAM levels. Luciferase reporter assays and chromatin immunoprecipitation indicated the functionality of TEAD2 site on TFAM promoter in mediating its YAP1-dependent expression. YAP1-dependent mitochondrial fragmentation and ROS generation was accompanied by decreased TERT levels and increased mitochondrial TERT localization in IDH1 R132H cells. Treatment with Bosutinib that prevents extranuclear TERT shuttle, further elevated ROS in IDH1 R132H cells and triggered apoptosis. Importantly, Bosutinib elevated ROS levels and induced apoptosis in IDH1 WT cells upon concurrent depletion of YAP1. These findings highlight the involvement of YAP1 in coupling mitochondrial dysfunction with TERT mitochondrial shuttle to constitute an essential non-canonical function of YAP1 in regulating redox homeostasis.

scholarly journals Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

2021 ◽  
Vol 22 (11) ◽  
pp. 5590
Author(s):  
Clément Veys ◽  
Abderrahim Benmoussa ◽  
Romain Contentin ◽  
Amandine Duchemin ◽  
Emilie Brotin ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Functional Screening ◽  
Western Blots ◽  
Egfr Expression ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis ◽  
First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

scholarly journals miR-125b Disrupts Mitochondrial Dynamics via Targeting Mitofusin 1 in Cisplatin-Induced Acute Kidney Injury

2021 ◽  
pp. 1-11
Author(s):  
Yue Zhao ◽  
Yue Lang ◽  
Mingchao Zhang ◽  
Shaoshan Liang ◽  
Xiaodong Zhu ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Molecular Mechanisms ◽  
Mitochondrial Dynamics ◽  
Kidney Injury ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Mitochondrial Fragmentation ◽  
Tubular Cells ◽  
Renal Tubular Cells

<b><i>Background:</i></b> Mitochondria are dynamic organelles whose structure are maintained by continuous fusion and fission. During acute kidney injury (AKI) progression, mitochondrial fission in renal tubular cells was elevated, characterized by mitochondrial fragmentation. It is tightly associated with mitochondrial dysfunction, which has been proven as a critical mechanism responsible for AKI. However, the initiating factor for the disruption of mitochondrial dynamics in AKI was not well understood. <b><i>Objectives:</i></b> To explore the molecular mechanisms of mitochondrial disorders and kidney damage. <b><i>Methods:</i></b> We established cisplatin-induced AKI model in C57BL/6 mice and proximal tubular cells, and detected the expression of miR-125b by qPCR. Then we delivered miR-125b antagomir after cisplatin treatment in mice via hydrodynamic-based gene transfer technique. Subsequently, we performed luciferase reporter and immunoblotting ­assays to prove miR-125b could directly modulate mitofusin1 (MFN1) expression. We also tested the role of miR-125b in mitochondrial and renal injury through immunofluorescent staining, qPCR, and immunoblotting assays. <b><i>Results:</i></b> miR-125b levels were induced in cisplatin-challenged mice and cultured tubular cells. Anti-miR-125b could effectively alleviate cisplatin-induced mitochondrial fragmentation and kidney injury both in vitro and in vivo. Furthermore, miR-125b could directly regulate MFN1, which is a key regulator of mitochondrial fusion. Our study indicated that miR-125b is upregulated during cisplatin-induced AKI. Inhibition of miR-125b may suppress mitochondrial and renal damage through upregulating MFN1. This study suggests that miR-125b could be a potential therapeutic target in AKI.

scholarly journals Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 290 ◽  
Author(s):  
Alagie Jassey ◽  
Ching-Hsuan Liu ◽  
Chun Changou ◽  
Christopher Richardson ◽  
Hsue-Yin Hsu ◽  
...  
Keyword(s):  
Mitochondrial Dynamics ◽  
Underlying Mechanism ◽  
Effector Protein ◽  
Autophagic Flux ◽  
Oxygen Species ◽  
Structural Protein ◽  
Mitochondrial Fragmentation ◽  
Lysosomal Degradation ◽  
Subgenomic Replicon ◽  
Important Regulator

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

scholarly journals miR-128 Targets the SIRT1/ROS/DR5 Pathway to Sensitize Colorectal Cancer to TRAIL-Induced Apoptosis

2018 ◽  
Vol 49 (6) ◽  
pp. 2151-2162 ◽  
Author(s):  
Bo Lian ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Gang Shi ◽  
Jibin Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal anti-tumor drug because it exhibits selective cytotoxicity against cancer cells. However, certain cancer cells are resistant to TRAIL, and the potential mechanisms are still unclear. The aim of this study was to reduce the resistance of colorectal cancer (CRC) cells to TRAIL. Methods: Quantitative real-time PCR analysis was performed to detect the expression of microRNA-128 (miR-128) in tissues from patients with CRC and CRC cell lines. MTT assays were used to evaluate the effect of miR-128 on TRAIL-induced cytotoxicity against CRC cell lines. The distribution of death receptor 5 (DR5) and the production of reactive oxygen species (ROS) were detected by flow cytometry analysis. Western blot, flow cytometry, and luciferase reporter assays were performed to evaluate the potential mechanism and pathway of miR-128-promoted apoptosis in TRAIL-treated CRC cells. Results: MiR-128 expression was downregulated in tumor tissues from patients with CRC as well as in CRC cell lines in vitro. The enforced expression of miR-128 sensitized CRC cells to TRAIL-induced cytotoxicity by inducing apoptosis. Mechanistically, bioinformatics, western blot analysis, and luciferase reporter assays showed that miR-128 directly targeted sirtuin 1 (SIRT1) in CRC cells. miR-128 overexpression suppressed SIRT1 expression, which promoted the production of ROS in TRAIL-treated CRC cells. This increase of ROS subsequently induced DR5 expression, and thus increased TRAIL-induced apoptosis in CRC cells. Conclusion: The combination of miR-128 with TRAIL may represent a novel approach for the treatment of CRC.

scholarly journals MicroRNA-185 induces potent autophagy via AKT signaling in hepatocellular carcinoma

Tumor Biology ◽  
2017 ◽  
Vol 39 (2) ◽  
pp. 101042831769431 ◽  
Author(s):  
Li Zhou ◽  
Shunai Liu ◽  
Ming Han ◽  
Shenghu Feng ◽  
Jinqiu Liang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepg2 Cells ◽  
Target Genes ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Promising Therapeutic Target ◽  
Reporter Assays ◽  
Induced Apoptosis ◽  
First Time

Studies have demonstrated that microRNA 185 may be a promising therapeutic target in liver cancer. However, its role in hepatocellular carcinoma is largely unknown. In this study, the proliferation of human HepG2 cells was inhibited by transfection of microRNA 185 mimics. Cell-cycle analysis revealed arrest at the G0/G1 phase. Transfection of HepG2 cells with microRNA 185 mimics significantly induced apoptosis. These data confirmed microRNA 185 as a potent cancer suppressor. We demonstrated that microRNA 185 was a compelling inducer of autophagy, for the first time. When cell autophagy was inhibited by chloroquine or 3-methyladenine, microRNA 185 induced more cell apoptosis. MicroRNA 185 acted as a cancer suppressor by regulating AKT1 expression and phosphorylation. Dual-luciferase reporter assays indicated that microRNA 185 suppressed the expression of target genes including RHEB, RICTOR, and AKT1 by directly interacting with their 3′-untranslated regions. Binding site mutations eliminated microRNA 185 responsiveness. Our findings demonstrate a new role of microRNA 185 as a key regulator of hepatocellular carcinoma via autophagy by dysregulation of AKT1 pathway.

scholarly journals Restore of miR-541 resensitizes pancreatic cancer cells to gemcitabine-induced apoptosis through suppression of HAX-1

2020 ◽  
Author(s):  
Hong Liu ◽  
Xuemei Gan ◽  
Jun Zhang ◽  
Xingdiao Zhang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Potential Strategy ◽  
Induced Apoptosis ◽  
Mtt Assays

Abstract Background: MiR-541 acts as a tumor suppressor in some cancers. However, the role of miR-541 in regulating the chemosensitivity to cancer cells is still unclear. The aim of this study is to explore the effect of miR-541 on chemoresistance of pancreatic cancer (PCa) cells to gemcitabine-induced apoptosis.Methods: Gemcitabine-resistant Panc-1 and Capan-2 PCa cell lines (Panc-1/R and Capan-2/R) were established through long term exposure to gemcitabine. Effect of miR-541 on changing the sensitivity of Panc-1/R and Capan-2/R to gemcitabine-induced cytotoxicity was evaluated by MTT assays. Regulation of miR-541 on HAX-1 was confirmed by bioinformatics, western blot analysis and luciferase reporter assays. Cell apoptosis and mitochondrial membrane potential (MMP) was measured by flow cytometry analysis.Results: Comparison with Panc-1 and Capan-2, downregulation of miR-541 was observed in Panc-1/R and Capan-2/R cells. Overexpression of miR-541 was found to increase the cytotoxicity of gemcitabine to Panc-1/R and Capan-2/R cells. However, transfection with HAX-1 plasmid can abolish the effect of miR-541 on gemcitabine-induced cytotoxicity against Panc-1/R and Capan-2/R.Conclusion: Downregulation of miR-541 is responsible for development of gemcitabine resistance in PCa. Overexpression of miR-541 may represent a potential strategy to reverse the chemoresistance of PCa.

scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

scholarly journals Functional Characterization of Melanocortin-3 Receptor In Rainbow Trout (Oncorhynchus Mykiss)

2021 ◽  
Author(s):  
Hui-Xia Yu ◽  
Yang Li ◽  
Wei-Jia Song ◽  
Hui Wang ◽  
Hao-Lin Mo ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Energy Homeostasis ◽  
Functional Characterization ◽  
Luciferase Reporter ◽  
Intracellular Camp ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Bony Fishes ◽  
Important Regulator ◽  
Reporter Assays

Abstract The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis and inflammation in mammals. However, its function in teleost fish needs to be further explored. In this study, we characterized rainbow trout MC3R (rtMC3R), which encoded a putative protein of 331 amino acids. Phylogenetic and chromosomal synteny analyses showed that rtMC3R was closely related to bony fishes. Quantitative PCR (qPCR) revealed that the transcripts of rtMC3R were highly expressed in the brain and muscle. The cellular function of rtMC3R was further verified by the signal-pathway-specific luciferase reporter assays. Four agonists such as α-MSH, β-MSH, ACTH (1–24) and NDP-MSH can active rtMC3R, increasing the production of intracellular cAMP and up-regulating MAPK/ERK signals. Moreover, we found that rtMC3R stimulated with α-MSH and NDP-MSH can significantly inhibit the NF-κB signaling pathway. This research will be helpful for further studies on the function of MC3R in rainbow trout, especially the role of energy metabolism and immune regulation.

scholarly journals circZMYM2 Competed Endogenously with miR-335-5p to Regulate JMJD2C in Pancreatic Cancer

2018 ◽  
Vol 51 (5) ◽  
pp. 2224-2236 ◽  
Author(s):  
Yong An ◽  
Huihua Cai ◽  
Yue Zhang ◽  
Shengyong Liu ◽  
Yunfei Duan ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Tissue Samples ◽  
Paired Normal Tissue ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: We aimed to study the involvement of circZMYM2 (hsa_circ_0099999) in pancreatic cancer (PC) cell proliferation, apoptosis and invasion and to figured out the underlying mechanism of circZMYM2 regulating miR-335-5p and JMJD2C. Methods: CircRNA differential expressions in twenty PC samples and paired normal tissue samples were analyzed using Arraystar Human CircRNA microarray V1. CircZMYM2 expression level was determined via qRT-PCR. The effects of circZMYM2 inhibition and overexpression on cell proliferation, cell apoptosis and cell invasion were investigated by CCK-8 assays, Flow cytometry assays and Transwell assays. An animal experiment on nude mice was put forward to test the influence of circZMYM2 knockdown on tumor growth. The relationship between circZMYM2, miR-335 and JMJD2C was verified by RNA pull down, dual-luciferase reporter assays and rescue experiment. The effect of circZMYM2 and miR-335-5p on the expression of JMJD2C protein was detected by western blot. Results: CircZMYM2 overexpression was observed in both PC tissues and cells. Knockdown of circZMYM2 inhibited proliferation, induced apoptosis, and weakened invasion ability of cancer cells. Tumor growth was restrained in vivo. CircZMYM2 repressed the expression of its target miR-335-5p. MiR-335-5p attenuated pancreatic cancer development via inhibition of JMJD2C. Conclusion: Our study demonstrated that circZMYM2 promoted PC progression. CircZMYM2 had a sponge effect on miR-335-5p and modulated the downstream oncogene JMJD2C.

Sign in / Sign up

Export Citation Format

Share Document