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 Molecular Cloning and Functional Characterization of Three 5-HT Receptor Genes (HTR1B, HTR1E and HTR1F) in Chickens

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 891
Author(s):  
Caiyun Sun ◽  
Yang Qiu ◽  
Qin Ren ◽  
Xiao Zhang ◽  
Baolong Cao ◽  
...  
Keyword(s):  
Serotonin Receptor ◽  
Functional Characterization ◽  
Brain Regions ◽  
Luciferase Reporter ◽  
Molecular Characteristics ◽  
Intracellular Camp ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Serotonin Signaling ◽  
Reporter Assays

The serotonin (5-hydroxytryptamine, 5-HT) signaling system is involved in a variety of physiological functions, including the control of cognition, reward, learning, memory, and vasoconstriction in vertebrates. Contrary to the extensive studies in the mammalian system, little is known about the molecular characteristics of the avian serotonin signaling network. In this study, we cloned and characterized the full-length cDNA of three serotonin receptor genes (HTR1B, HTR1E and HTR1F) in chicken pituitaries. Synteny analyses indicated that HTR1B, HTR1E and HTR1F were highly conserved across vertebrates. Cell-based luciferase reporter assays showed that the three chicken HTRs were functional, capable of binding their natural ligands (5-HT) or selective agonists (CP94253, BRL54443, and LY344864) and inhibiting intracellular cAMP production in a dose-dependent manner. Moreover, activation of these receptors could stimulate the MAPK/ERK signaling cascade. Quantitative real-time PCR analyses revealed that HTR1B, HTR1E and HTR1F were primarily expressed in various brain regions and the pituitary. In cultured chicken pituitary cells, we found that LY344864 could significantly inhibit the secretion of PRL stimulated by vasoactive intestinal peptide (VIP) or forskolin, revealing that HTR1F might be involved in the release of prolactin in chicken. Our findings provide insights into the molecular mechanism and facilitate a better understanding of the serotonergic modulation via HTR1B, HTR1E and HTR1F in avian species.

scholarly journals MITF induces escape from innate immunity in melanoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Luis Sánchez-del-Campo ◽  
Román Martí-Díaz ◽  
María F. Montenegro ◽  
Rebeca González-Guerrero ◽  
Trinidad Hernández-Caselles ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Nk Cells ◽  
Nk Cell ◽  
Luciferase Reporter ◽  
Damage Repair ◽  
Reporter Assays ◽  
Underlying Mechanisms ◽  
Nk Cytotoxicity

Abstract Background The application of immune-based therapies has revolutionized cancer treatment. Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood. In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair. Whether MITF also controls the immune response is unknown. Methods By using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response. ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene. Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing. Results Here we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells. By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system. Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance. Conclusion Our results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.

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 The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta)

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Shihang Sun ◽  
Chungen Hu ◽  
Xiujuan Qi ◽  
Jinyong Chen ◽  
Yunpeng Zhong ◽  
...  
Keyword(s):  
Cold Stress ◽  
Freezing Tolerance ◽  
Luciferase Reporter ◽  
Dna Interactions ◽  
Functional Protein ◽  
Actinidia Arguta ◽  
Protein Dna Interactions ◽  
Similar Phenotype ◽  
Reporter Assays

AbstractBeta-amylase (BAM) plays an important role in plant resistance to cold stress. However, the specific role of the BAM gene in freezing tolerance is poorly understood. In this study, we demonstrated that a cold-responsive gene module was involved in the freezing tolerance of kiwifruit. In this module, the expression of AaBAM3.1, which encodes a functional protein, was induced by cold stress. AaBAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance, and the heterologous overexpression of AaBAM3.1 in Arabidopsis thaliana resulted in a similar phenotype. The results of promoter GUS activity and cis-element analyses predicted AaCBF4 to be an upstream transcription factor that could regulate AaBAM3.1 expression. Further investigation of protein-DNA interactions by using yeast one-hybrid, GUS coexpression, and dual luciferase reporter assays confirmed that AaCBF4 directly regulated AaBAM3.1 expression. In addition, the expression of both AaBAM3.1 and AaCBF4 in kiwifruit responded positively to cold stress. Hence, we conclude that the AaCBF-AaBAM module is involved in the positive regulation of the freezing tolerance of kiwifruit.

scholarly journals Hsa_circ_0026628 promotes the development of colorectal cancer by targeting SP1 to activate the Wnt/β-catenin pathway

2021 ◽  
Vol 12 (9) ◽  
Author(s):  
Xuexiu Zhang ◽  
Jianning Yao ◽  
Haoling Shi ◽  
Bing Gao ◽  
Haining Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcription Factor ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Transcriptional Level ◽  
Sp1 Transcription Factor ◽  
Reporter Assays ◽  
Sphere Formation

AbstractCircular RNAs (circRNAs) have been reported to play crucial roles in the progression of various cancers, including colorectal cancer (CRC). SP1 (Sp1 transcription factor) is a well-recognized oncogene in CRC and is deemed to trigger the Wnt/β-catenin pathway. The present study was designed to investigate the role of circRNAs which shared the same pre-mRNA with SP1 in CRC cells. We identified that hsa_circ_0026628 (circ_0026628), a circular RNA that originated from SP1 pre-mRNA, was upregulated in CRC cells. Sanger sequencing and agarose gel electrophoresis verified the circular characteristic of circ_0026628. Functional assays including CCK-8, colony formation, transwell, immunofluorescence staining, and sphere formation assay revealed the function of circ_0026628. RNA pull-down and mass spectrometry disclosed the proteins interacting with circ_0026628. Mechanistic assays including RIP, RNA pull-down, CoIP, ChIP, and luciferase reporter assays demonstrated the interplays between molecules. The results depicted that circ_0026628 functioned as a contributor to CRC cell proliferation, migration, EMT, and stemness. Mechanistically, circ_0026628 served as the endogenous sponge of miR-346 and FUS to elevate SP1 expression at the post-transcriptional level, thus strengthening the interaction between SP1 and β-catenin to activate the Wnt/β-catenin pathway. In turn, the downstream gene of Wnt/β-catenin signaling, SOX2 (SRY-box transcription factor 2), transcriptionally activated SP1 and therefore boosted circ_0026628 level. On the whole, SOX2-induced circ_0026628 sponged miR-346 and recruited FUS protein to augment SP1, triggering the downstream Wnt/β-catenin pathway to facilitate CRC progression.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

scholarly journals The role of nitrosative and oxidative stress in rainbow trout (Oncorhynchus mykiss) liver tissue applied mercury chloride (HgCl2)

2021 ◽  
Vol 38 (3) ◽  
pp. 269-273
Author(s):  
Mehmet Reşit Taysı ◽  
Muammer Kırıcı ◽  
Mahinur Kırıcı ◽  
Hasan Ulusal ◽  
Bünyamin Söğüt ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Liver Tissue ◽  
Stress Index ◽  
Mercury Chloride ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Total Antioxidant ◽  
Ld50 Value

The aim of this study was to determine oxidative stress caused by mercury chloride (HgCl2) in rainbow trout (Oncorhynchus mykiss) liver tissue. For this purpose, the LD50 value of HgCl2 on rainbow trout was determined as 551 μg/L. In the study, 40 fish in four groups were exposed to 25% and 50% (138 and 276 µg/L) of the two subletal doses of HgCl2 for 2 and 7 days, with 10 fish (n=10) in each group. To determine oxidative stress; peroxynitrite (ONOO−), total oxidant level (TOS), total antioxidant level (TAS), oxidative stress index (OSI) and malondialdehyde (MDA) were analyzed. In the study, it was observed that the differences between the groups in terms of ONOO−, TOS, TAS and OSI levels in the liver tissues was significant (P<0.05), however, this difference was not significant (P>0.05) in terms of MDA values. As a result, it can be concluded that HgCl2 increases ONOO−, TOS, TAS, OSI and MDA levels in liver tissue and even small doses of mercury are toxic to fish.

scholarly journals Chorionic Villous Mesenchymal Stem Cells Derived Exosomes Deliver miR-135b-5p to Trophoblasts, Promoting their Proliferation and Invasion by Targeting TXNIP via β-Catenin Pathway

2020 ◽  
Author(s):  
Yijing Chu ◽  
Yan Zhang ◽  
Guoqiang Gao ◽  
Jun Zhou ◽  
Yang Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tissue Injury ◽  
Luciferase Reporter ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Reporter Assays ◽  
Pcr Assays ◽  
Proliferation And Invasion

Abstract Background: Human chorionic villous mesenchymal stem cells (CV-MSCs) are found to be a promising and effective treatment for tissue injury. Trophoblast dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work was to understand how CV-MSCs regulated trophoblast function. Methods: In this study, we treated trophoblasts with CV-MSC-derived exosomes and RNA-seq analysis was used to understand the changes in trophoblasts. We examined the levels of TXNIP and β-catenin in trophoblasts by immunohistochemistry, western blot and qRT-PCR assays. Luciferase reporter assays and qRT-PCR assays were used to understand the role of miR135b-5p in the effects of CV-MSC-derived exosomes. The growth and invasion of trophoblasts was evaluated with the CCK-8 and transwell assays. Results: The treatment markedly enhanced the trophoblast proliferation and invasion. Furthermore, a significant decrease of TXNIP expression and inactivation of the β-catenin pathway in CV-MSCs exosomes-treated trophoblasts was observed. Consistent with these findings, TXNIP inhibition exhibited the same effect of promoting trophoblast proliferation and invasion as induced by CV-MSC-derived exosomes, also with the accompaniment of inactivation of β-catenin pathway. In addition, overexpression of TXNIP activated the β-catenin pathway in trophoblasts, and reduced the proliferation and invasion of trophoblasts. Importantly, miR135b-5p was found to be highly expressed in CV-MSC exosomes and interact with TXNIP. The miR-135b-5p overexpression significantly elevated the proliferation and invasion of trophoblasts, which could be attenuated by TXNIP overexpression. Conclusion: Our results suggest that TXNIP-dependent β-catenin pathway inactivation mediated by miR135b-5p which is delivered by CV-MSC-derived exosomes could promote the proliferation and invasion of trophoblasts.

scholarly journals MiR-125b-5p Inhibited The Progression of Hepatoblastoma As A Shared miRNA of The lncRNA NEAT1 and YES1

2020 ◽  
Author(s):  
Zhu Jin ◽  
Yutong Chen ◽  
Yuchen Mao ◽  
Mingjuan Gao ◽  
Zebing Zheng ◽  
...  
Keyword(s):  
Clinicopathological Characteristics ◽  
Luciferase Reporter ◽  
Tumor Growth Inhibition ◽  
Invasion And Migration ◽  
In Vivo Experiments ◽  
Reporter Assays ◽  
And Migration ◽  
Relationship Of

Abstract Background: microRNAs have been studied widely in hepatoblastoma. However, the role of miR-125b-5p and its relationship with the lncRNA sNEAT1 and YES1 in hepatoblastoma have not been reported previously. We aimed to reveal the role of NEAT1/miR-125b-5p/YES1 in the progression of hepatoblastoma.Methods: We collected tumor tissues and their adjacent tissues from 12 hepatoblastoma patients. qRT-PCR was applied to detect the expression of miR-125b-5p, and the relationship of miR-125b-5p with clinicopathological characteristics was analyzed. Dual luciferase reporter assays and RNA pull down assays were used to identify the relationships among NEAT1, miR-125b-5p and YES1. CCK8, Transwell assays and wound healing assays were used to examine cell viability, invasion and migration. In vivo experiments were also applied to detect the effect of miR-125b-5p on hepatoblastoma.Results: miR-125b-5p was significantly downregulated in hepatoblastoma tissue and cells. The higher the PRETEXT grade, the lower the miR-125b-5p level. NEAT1 could bind to miR-125b-5p and inhibit its expression. miR-125b-5p could target YES1 and inhibit its expression. Overexpression of miR-125b-5p decreased the proliferation, invasion, and migratory ability of hepatoblastoma cells. YES1 could rescue the above effects. At the same time, overexpression of miR-125b-5p resulted in decreased YES1 and tumor growth inhibition in vivo.Conclusion: miR-125b-5p acted as a shared miRNA of NEAT1 and YES1 in hepatoblastoma. Overexpression of miR-125b-5p could target YES1 and inhibit its expression, therefore inhibiting the progression of hepatoblastoma.

Sign in / Sign up

Export Citation Format

Share Document