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 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 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.

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response

2021 ◽  
Author(s):  
Zemin Wang ◽  
Darren Chern Jan Wong ◽  
Yi Wang ◽  
Guangzhao Xu ◽  
Chong Ren ◽  
...  
Keyword(s):  
Stress Response ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Cold Treatment ◽  
Ectopic Expression ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Vitis Amurensis ◽  
Mobility Shift

Abstract Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

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 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.

AK048794 maintains the mouse embryonic stem cell pluripotency by functioning as an miRNA sponge for miR-592

2016 ◽  
Vol 473 (20) ◽  
pp. 3639-3654 ◽  
Author(s):  
Yang Zhou ◽  
Qing-Song Dai ◽  
Shi-Chang Zhu ◽  
Yue-Hua Han ◽  
Hai-Long Han ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Luciferase Reporter ◽  
Stem Cell Pluripotency ◽  
Reporter Assays ◽  
Pluripotency Maintenance ◽  
Embryonic Stem Cell Pluripotency

MiR-592 has been identified as a neural-enriched microRNA, plays an important role in mNPCs differentiation, could induce astrogliogenesis differentiation arrest or/and enhance neurogenesis in vitro. Previous studies showed that long noncoding RNAs (lncRNAs) were involved in the neuronal development and activity. To investigate the role of miR-592 in neurogenesis, we described the expression profile of lncRNAs in miR-592 knockout mouse embryonic stem cells (mESCs) and the corresponding normal mESCs by microarray. By the microarray analysis and luciferase reporter assays, we demonstrated that lncRNA - AK048794, regulated by transcription factor GATA1, functioned as a competing endogenous RNA (ceRNA) for miR-592 and led to the de-repression of its endogenous target FAM91A1, which is involved in mESC pluripotency maintenance. Taken together, these observations imply that AK048794 modulated the expression of multiple genes involved in mESC pluripotency maintenance by acting as a ceRNA for miR-592, which may build up the link between the regulatory miRNA network and mESC pluripotency.

scholarly journals The Mechanisms of Colorectal Cancer Cell mesenchymal-epithelial Transition Induced by Hepatocyte Exosome-derived miR-203a-3p

2021 ◽  
Author(s):  
Heyang Xu ◽  
Qiusheng Lan ◽  
Yongliang Huang ◽  
Yang Zhang ◽  
Yujie Zeng ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Real Time Quantitative Pcr ◽  
Mesenchymal To Epithelial Transition ◽  
Reporter Assays ◽  
Phosphatase Of Regenerating Liver

Abstract Background: Liver metastasis is the most common cause of death in patients with colorectal cancer (CRC). Phosphatase of regenerating liver-3 induces CRC metastasis by epithelial-to-mesenchymal transition, which promotes CRC cell liver metastasis. Mesenchymal-to-epithelial transition (MET), the opposite of epithelial-to-mesenchymal transition, has been proposed as a mechanism for the establishment of metastatic neoplasms. However, the molecular mechanism of MET remains unclear. Methods: Using Immunohistochemistry, western blotting，invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, human miRNA arrays, and xenograft mouse model, we determined the role of hepatocyte exosome-derived miR-203a-3p in CRC MET.Results: In our study, we found that miR-203a-3p derived from hepatocyte exosomes increased colorectal cancer cells E-cadherin expression, inhibited Src expression, and reduced activity. In this way miR-203a-3p induced the decreased invasion rate of CRC cells.Coclusion: MiR-203a-3p derived from hepatocyte exosomes plays an important role of CRC cells to colonize in liver.

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