scholarly journals Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1532 ◽  
Author(s):  
Ning Li ◽  
Joachim F. Uhrig ◽  
Corinna Thurow ◽  
Li-Jun Huang ◽  
Christiane Gatz
Keyword(s):  
Signaling Pathway ◽  
Function Analysis ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Activation Of Transcription ◽  
Subsequent Degradation ◽  
The Stability ◽  
F Box Protein ◽  
Ja Signaling

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aos coi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

scholarly journals Overexpressed microrna-129-5p reverses CCl4-Induced hepatic fibrosis by suppressing PEG3 expression and the NF-κB signaling pathway

2020 ◽  
Author(s):  
Shengtao Sun ◽  
Yunxia Ma ◽  
Yinfeng Li
Keyword(s):  
Signaling Pathway ◽  
Hepatic Fibrosis ◽  
Gene Expression Regulation ◽  
Function Analysis ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Related Factors ◽  
Gene Assay

AbstractHepatic fibrosis is a pathological process resulting from liver damage, which leads to the extracellular matrix (ECM) proteins accumulation in the liver. Considering that microRNA (miR)-129-5p has a vital effect in the gene expression regulation about fibrosis through transcriptional profiling, this study speculated whether miR-129-5p had potential to influence the progression of hepatic fibrosis. The hepatic fibrosis rat models induced by C-C motif chemokine ligand 4 (CCl4) were established. The pathological changes of the liver tissues were assayed with hematoxylin-eosin (HE) staining. Subsequently, gain- and loss-of-function analysis with miR-129-5p antagomir or shRNA against PEG3 was conducted to further investigate the molecular regulatory mechanism of miR-129-5p, with detection of the expression of NF-κB signaling pathway-related proteins and apoptosis-related factors. The serum samples of rats were analyzed by serological index analysis. The targeting of miR-129-5p to PEG3 was verified by dual-luciferase reporter gene assay. The detection of apoptosis in rats was measured by TUNEL staining. MiR-129-5p was poorly-expressed and PEG3 was highly-expressed in hepatic fibrosis. miR-129-5p could reduce the expression of PEG3. Next, upregulated miR-129-5p or downregulated PEG3 led to less obvious histological changes of liver cirrhosis and lowered apoptosis rate. Further, miR-129-5p regulated the activation of NF-κB signaling pathway via PEG3. The hepatic fibrosis induced by CCl4 can be reversed by upregulated miR-129-5p or downregulated PEG3 expression.

scholarly journals miRNA-331-3p Affects The Proliferation, Metastasis And Invasion of Osteosarcoma Through SOCS1/JAK2/STAT3

2021 ◽  
Author(s):  
Dan Zu ◽  
Qi Dong ◽  
Sunfang Chen ◽  
Yongde Chen ◽  
Jun Yao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Small Noncoding Rnas ◽  
Protein Levels ◽  
Stat3 Signaling ◽  
Survival Difference ◽  
Stat3 Signaling Pathway

Abstract Background: MicroRNAs (miRNA) are regulatory small noncoding RNAs, which play a key role in many cancers. It has been found that miR-331-3p is involved in the development and progression of various cancers, but there are few reports in osteosarcoma. Methods: The public GEO database was used to analyze the survival difference of miR-331-3p in OS organizations. The level of cell proliferation assay was assessed by CCK-8 and colony formation. Tanswell and Wound-healing detect the transfer and invasion ability of miR-331-3p in OS. TargetScan, miRDBmiR, TarBase, and dual luciferase reporter gene assays were used to determine SOCS1 as a targeted regulator. Western blot and immunohistochemistry were used to detect the expression of protein levels. A mouse model of subcutaneously transplantable tumors is used to evaluate the proliferation of OS in vivo.Results: The low expression of miR-331-3p is negatively correlated with the overall survival of OS patients. Overexpression of miR-331-3p significantly inhibited cell proliferation, metastasis and invasion. miR-331-3p affects the occurrence and development of osteosarcoma by targeting the SOCS1/JAK2/STAT3 signaling pathway.Conclusion: miR-331-3p reduces the expression of SOCS1 by combining with its 3'UTR, thereby activating the JAK2/STAT3 signaling pathway to regulate the progression of OS.

scholarly journals Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

Pharmaceutics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 62
Author(s):  
Mangesh Morey ◽  
Akshay Srivastava ◽  
Abhay Pandit
Keyword(s):  
Gene Delivery ◽  
Fold Increase ◽  
Nonviral Gene Delivery ◽  
Luciferase Reporter ◽  
Hydrodynamic Diameter ◽  
Luciferase Reporter Gene ◽  
One Pot ◽  
Gaussia Luciferase ◽  
Nih3t3 Cells ◽  
The Stability

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in 50 mM glucose compared to that of 10 mM glucose.

Overexpressed miR-375-Loaded Restrains Development of Cervical Cancer Through Down-Regulation of Frizzled Class Receptor 4 (FZD4) with Liposome Nanoparticle as a Carrier

2021 ◽  
Vol 17 (9) ◽  
pp. 1882-1889
Author(s):  
Suqin Wang ◽  
Lina Xu ◽  
Zhiqiang Zhang ◽  
Ping Wang ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
M Phase ◽  
The Impact

Dysregulation expression of miR-375 is noted to correlate with progression of cervical cancer. This study attempted to investigate the impact of overexpressed miR-375-loaded liposome nanoparticles on proliferation of cervical cancer (CC), to provide an insight on pathogenesis of CC disorder. CC cells were co-cultured with pure liposome nanoparticles (empty vector group), miR-375 agonist-loaded liposome nanoparticles, or transfected with miR-375 antagonist. Besides, some cells were exposed to TGF-β/Smads signaling pathway inhibitor or activator whilst cell proliferation was assessed by MTT assay, and expressions of FZD4 and miR-375 were determined. Western blot analysis was carried out to detect the expression of TGF-β pathway factors (TGF-β, Smad2, Smad7, p-Smad2) and its downstream Smads pathway. The interaction between miR-375 and FZD4 was evaluated by dual-luciferase reporter gene assay. Overexpression of miR-375 induced arrest at the G0/G1 phase of cell cycle and elevation of Smad2 protein expression (P <0.05), with lower expressions of TGF-β, Smad7, p-Smad2, and FZD4, while transfection with miR-375 inhibitor exhibited opposite activity. Presence of miR-375 agonist-loaded liposome nanoparticles induced decreased cell proliferation. There was a targeting relationship between miR-375 and FZD4, and administration with TGF-β/Smads agonist resulted in increased miR-375 and Smad2 expressions, as well as decreased TGF-β, Smad7, p-Smad2, FZD4 protein expression, and the number of S phase and G2/M phase cells (P < 0.05). The signaling inhibitor oppositely suppressed cell proliferation decreasing miR-375 expression. miR-375-loaded liposome nanoparticles activated TGF-β/Smads signaling pathway to restrain cell cycle and suppress cell division, and proliferation through targeting FZD4 in CC. Its molecular mechanism is related to activation of TGF-β/Smads signaling pathway.

scholarly journals LncRNA HCG11 promotes proliferation and migration in gastric cancer via targeting miR-1276/CTNNB1 and activating Wnt signaling pathway

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Hua Zhang ◽  
Haitao Huang ◽  
Xiaomei Xu ◽  
Haiying Wang ◽  
Jianxiang Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Function Analysis ◽  
Wnt Signaling Pathway ◽  
Tumor Formation ◽  
Tumor Promoter ◽  
Luciferase Reporter ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Gastric cancer (GC) is one common cancer which occurs in the stomach leading to high mortality around the world. Long non-coding RNAs (lncRNAs) were found overexpressed or silenced in the occurrence and progression of multiple cancers including GC. Method The gene expression level in GC tissues and cells were analyzed by RT-qPCR. CCK-8, colony formation, flow cytometry and transwell assays were performed for the function analysis of HLA complex group 11 (HCG11). The mechanism study for HCG11 was conducted using RIP, RNA pull down and luciferase reporter assays. Results HCG11 was discovered highly expressed in GC tissues and cells. Depletion experiments were used to evaluate HCG11 silence on cell proliferation, migration and apoptosis. Moreover, Wnt signaling pathway was found as a tumor promoter in GC. RIP assay, RNA pull down assay and luciferase reporter assay were performed to illustrate the relationship of HCG11, miR-1276 and CTNNB1. Rescue assays revealed that HCG11/miR-1276/CTNNB1 axis regulated the incidence and development of GC. Tumor formation in mice proved that HCG11 was negatively correlated with miR-1276 and had positively correlation with CTNNB1. Conclusion Overall, HCG11 accelerated proliferation and migration in GC through miR-1276/CTNNB1 and Wnt signaling pathway, revealing that HCG11 could be a brand new target for GC.

scholarly journals Downregulation of microRNA-149 in retinal ganglion cells suppresses apoptosis through activation of the PI3K/Akt signaling pathway in mice with glaucoma

2018 ◽  
Vol 315 (6) ◽  
pp. C839-C849 ◽  
Author(s):  
Xin-Gang Nie ◽  
Dong-Sheng Fan ◽  
Yan-Xia Huang ◽  
Ying-Ying He ◽  
Bo-Li Dong ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Ultrastructural Changes ◽  
Retinal Ganglion ◽  
Akt Signaling Pathway ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Glaucoma represents a major cause of blindness, generally associated with elevated intraocular pressure (EIOP). The aim of the present study was to investigate whether microRNA-149 (miR-149) affects retinal ganglion cells (RGCs) and the underlying mechanism based on a mouse model of chronic glaucoma with EIOP. The successfully modeled mice were administered with mimics or inhibitors of miR-149. Next, the number of RGCs, ultrastructural changes of RGCs, and purity of RGCs in the retinal tissues were detected. Moreover, the RGCs were collected and subsequently treated with 60 mmHg pressure and transfected with a series of plasmids aiding in the regulation of the expression of miR-149 and betacellulin (BTC). The levels of miR-149, BTC, phosphatidylinositol 3-kinase (PI3K), and Akt were subsequently determined. Finally, RGC viability and apoptosis were detected accordingly. Dual luciferase reporter gene assay provided validation, highlighting BTC was indeed a target gene of miR-149. The downregulation of miR-149 is accompanied by an increased number of RGCs and decreased ultrastructural RGC alterations. Additionally, downregulated miR-149 was noted to increase the levels of BTC, PI3K, and Akt in both the retinal tissues and RGCs, whereas the silencing of miR-149 was observed to promote the viability of RGC and inhibit RGC apoptosis. Taken together, the results of the current study provided validation suggesting that the downregulation of miR-149 confers protection to RGCs by means of activating the PI3K/Akt signaling pathway via upregulation of BTC in mice with glaucoma. Evidence presented indicated the promise of miR-149 inhibition as a potential therapeutic strategy for glaucoma treatment.

scholarly journals VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

2020 ◽  
Vol 295 (49) ◽  
pp. 16826-16839
Author(s):  
Haifeng Zhang ◽  
Xiaozhi Rong ◽  
Caixia Wang ◽  
Yunzhang Liu ◽  
Ling Lu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Stability ◽  
Signaling Pathway ◽  
Cultured Cells ◽  
Critical Role ◽  
Family Members ◽  
Von Hippel Lindau ◽  
T Cell Factor ◽  
Protein Levels ◽  
The Stability

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Urea-associated oxidative stress and Gadd153/CHOP induction

1999 ◽  
Vol 276 (5) ◽  
pp. F786-F793 ◽  
Author(s):  
Zheng Zhang ◽  
Xiao-Yan Yang ◽  
David M. Cohen
Keyword(s):  
Oxidative Stress ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Kidney Medulla ◽  
Urea Treatment ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Mrna And Protein Expression ◽  
Reporter Gene Analysis

Urea treatment (100–300 mM) increased expression of the oxidative stress-responsive transcription factor, Gadd153/CHOP, at the mRNA and protein levels (at ≥4 h) in renal medullary mIMCD3 cells in culture, whereas other solutes did not. Expression of the related protein, CCAAT/enhancer-binding protein (C/EBP-β), was not affected, nor was expression of the sensor of endoplasmic reticulum stress, grp78. Urea modestly increased Gadd153 transcription by reporter gene analysis but failed to influence Gadd153 mRNA stability. Importantly, upregulation of Gadd153 mRNA and protein expression by urea was antioxidant sensitive. Accordingly, urea treatment was associated with oxidative stress, as quantitated by intracellular reduced glutathione content in mIMCD3 cells. In addition, antioxidant treatment partially inhibited the ability of urea to activate transcription of an Egr-1 luciferase reporter gene. Therefore oxidative stress represents a novel solute-signaling pathway in the kidney medulla and, potentially, in other tissues.

microRNA-422a Inhibits DCC Expression in a Manner Dependent on SNP rs12607853

2020 ◽  
Vol 160 (2) ◽  
pp. 63-71
Author(s):  
Yunxiao Li ◽  
Xugang Shi ◽  
Xintong Cai ◽  
Yongsheng Zhu ◽  
Yuanyuan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Opioid Addiction ◽  
Heroin Addiction ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay ◽  
New Biomarkers ◽  
And Function

DCC netrin 1 receptor (DCC) affects the structure and function of the dopamine circuitry, which in turn affects the susceptibility to developing addiction. In a previous study, we found that single nucleotide polymorphism (SNP) rs12607853 in the 3′ untranslated region (3′-UTR) of DCC was significantly associated with heroin addiction. In the current study, we first used bioinformatics prediction to identify the DCC rs12607853 C allele as a potential hsa-miR-422a and hsa-miR-378c target site. We then used vector construction and dual-luciferase reporter assays to investigate the targeting relationship of DCC rs12607853 with hsa-miR-422a and hsa-miR-378c. The dual-luciferase reporter gene assay confirmed that the C allele of rs12607853 in combination with hsa-miR-422a led to repressed dual-luciferase gene expression. Moreover, gene expression assays disclosed that hsa-miR-422a inhibited DCC expression at both the mRNA and protein levels. We also found that morphine inhibited the expression of hsa-miR-422a but increased the expression of DCC mRNA, and this change in the expression of hsa-miR-422a could not be reversed by naloxone, which suggested that the role of DCC in opioid addiction might be regulated by hsa-miR-422a. In summary, this study improves our understanding of the role of hsa-miR-422a and identifies the genetic basis of rs12607853, which might contribute to the discovery of new biomarkers or therapeutic targets for opioid addiction.

scholarly journals p54nrb Is a Transcriptional Corepressor of the Progesterone Receptor that Modulates Transcription of the Labor-Associated Gene, Connexin 43 (Gja1)

2009 ◽  
Vol 23 (8) ◽  
pp. 1147-1160 ◽  
Author(s):  
Xuesen Dong ◽  
Celeste Yu ◽  
Oksana Shynlova ◽  
John R. G. Challis ◽  
Paul S. Rennie ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Rna Splicing ◽  
Connexin 43 ◽  
Contractile Activity ◽  
Luciferase Reporter ◽  
Activator Protein 1 ◽  
Transcriptional Corepressor ◽  
Luciferase Reporter Gene ◽  
Dynamic Interactions ◽  
Protein Levels

Abstract The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.

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