scholarly journals m(6)A mRNA Methylation Regulates Ferroptosis in HPSCC by Targeting NFE2L2/NRF2

2021 ◽  
Author(s):  
Jing Ye ◽  
Xiaozhen Chen ◽  
Bing Liao ◽  
Xiaohua Jiang ◽  
Zhihuai Dong ◽  
...  
Keyword(s):  
Opposite Effect ◽  
Bioinformatics Analysis ◽  
Luciferase Reporter ◽  
Dot Blot ◽  
Transcriptional Inhibition ◽  
Reporter Assays ◽  
Nrf2 Expression ◽  
Insight Into ◽  
Dependent Mechanism

Abstract Background Emerging as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6-methyladenosine (m6A) modification has gathered tremendous scientific interest in recent years. However, no study addresses the role of m6A modification in ferroptosis. Here, we showed that m6A modifications are decreased in RSL3-induced ferroptosis in hypopharyngeal squamous cell carcinoma (HPSCC). We found that AlkB homolog 5 (ALKBH5), one of the m6A demethylases, is the primary factor involved in aberrant m6A modification. Methods Bioinformatics analysis, sample analysis, cell biological analyses and transcriptome sequencing were performed to evaluate the correlation between m6A modification and ferroptosis as well as molecular mechanism of ALKBH5 function. Transcriptome-wide m6A-seq and RIP-seq data and following m6A dot blot, MeRIP-qPCR, RIP-qPCR and dual luciferase reporter assays were mapped to screen and validate the candidate targets of ALKBH5. Results ALKBH5-knockdown impaired ferroptotic cell death in HPSCC. However, overexpression of ALKBH5 has an opposite effect, suggesting that ALKBH5 is a positive regulator of ferroptosis. Mechanistically, ALKBH5-mediated m6A demethylation led to a post-transcriptional inhibition of NFE2L2/NRF2, the central player in the regulation of antioxidant molecules in cells, at two m6A residues in the 3ʹ-UTR. Therefore, knocking down ALKBH5 subsequently increases the expression levels of NFE2L2/NRF2 and increased cell resistance to ferroptosis. In addition, m6A-mediated NFE2L2/NRF2 stabilization relied on the m6A reader IGF2BP2. Conclusion ALKBH5 functions as a tumor suppresser through ferroptosis in HPSCC. ALKBH5 destabilizes NFE2L2/NRF2 expression in HPSCC through an m6A-IGF2BP2-dependent mechanism. Together, our work uncovers a critical link between ALKBH5-NFE2L2/NRF2 and ferroptosis, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in HPSCC.

scholarly journals Role of MiR-98 and Its Underlying Mechanisms in Systemic Lupus Erythematosus

2018 ◽  
Vol 45 (10) ◽  
pp. 1397-1405 ◽  
Author(s):  
Lin Xie ◽  
Jinhua Xu
Keyword(s):  
T Cells ◽  
Lupus Erythematosus ◽  
Cd4 T Cells ◽  
Opposite Effect ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Fas Mrna ◽  
Reporter Assays ◽  
Underlying Mechanisms

Objective.T-lymphocyte apoptosis plays a critical role in the pathogenesis of systemic lupus erythematosus (SLE). However, the underlying regulatory mechanisms of apoptosis in SLE remain unclear. The aim of this study was to explore the role of miR-98 in SLE and its underlying mechanisms.Methods.Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to analyze miR-98 and Fas expression. Luciferase reporter assays were performed to identify miR-98 targets. To modify miRNA levels, miR-98 mimics and inhibitor were transfected into cells. A lentiviral construct was used to overexpress the level of Fas in SLE CD4+ T cells. Gene and protein expression were determined by qRT-PCR and Western blotting. Apoptosis levels were evaluated by annexin V staining and flow cytometry.Results.Compared to those of healthy donors, miR-98 was downregulated in SLE CD4+ T cells, whereas Fas mRNA and protein expression were upregulated. Upregulation of miR-98 by mimic transfection protected Jurkat cells against Fas-mediated apoptosis at both mRNA and protein levels, while miR-98 inhibitor induced the completely opposite effect. Luciferase reporter assays demonstrated that miR-98 directly targeted Fas mRNA. Further, miR-98 inhibitor induced apoptosis in primary healthy CD4+ T cells through the Fas-caspase axis, while upregulation of miR-98 in SLE CD4+ T cells led to the opposite effect.Conclusion.The current study revealed that downregulation of miR-98 induces apoptosis by modulating the Fas-mediated apoptotic signaling pathway in SLE CD4+ T cells. These results suggest that miR-98 might serve as a potential target for SLE treatment.

scholarly journals MicroRNA-152 Promotes Slow-Twitch Myofiber Formation via Targeting Uncoupling Protein-3 Gene

Animals ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 669 ◽  
Author(s):  
Yong Zhang ◽  
Honglin Yan ◽  
Pan Zhou ◽  
Zhenzhen Zhang ◽  
Jingbo Liu ◽  
...  
Keyword(s):  
Opposite Effect ◽  
Uncoupling Protein ◽  
Luciferase Reporter ◽  
Transcriptional Inhibition ◽  
Type Transformation ◽  
Uncoupling Protein 3 ◽  
Rescue Experiment ◽  
Slow Twitch ◽  
Myofiber Type

The differences of pork quality characteristics among different pig breeds mainly came from the differences in myofiber type compositions. Growing evidence indicated the key role of miRNAs in myofiber specification. In the present study, we found that miR-152 is more abundant in the slow-twitch myofiber-enriched muscles. However, its role in myofiber type transformation and myogenesis is largely unknown. Overexpression of miR-152 in porcine myotubes promoted the formation of slow-twitch myofibers and myogenesis. While, inhibition of miR-152 expression showed the opposite effect to miR-152 mimics transfection. The luciferase reporter analysis confirmed that miR-152 straightly targets the 3′-untranslated region (3’-UTR) of uncoupling protein 3 (UCP3) to cause its post-transcriptional inhibition in the protein level. The knockdown of UCP3 by siRNA showed the similar effect of miR-152 on myofiber type transition. Furthermore, the rescue experiment in the porcine myotube transfected with miR-152 mimics or/and UCP3 overexpression plasmid with or without the 3’UTR revealed that UCP3 mediates the action of miR-152 in slow-twitch myofiber formation. Taken together, our findings proposed a novel molecular mechanism through which miR-152 epigenetically regulates meat quality via promoting slow-twitch myofiber formation and skeletal myogenesis.

scholarly journals LncRNA SNHG16 Functions as an Oncogene by Sponging MiR-4518 and Up-Regulating PRMT5 Expression in Glioma

2018 ◽  
Vol 45 (5) ◽  
pp. 1975-1985 ◽  
Author(s):  
Ye-Fen Lu ◽  
Xue-Li Cai ◽  
Zheng-Zheng Li ◽  
Jing Lv ◽  
Yi-an Xiang ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Biological Function ◽  
Luciferase Reporter ◽  
Clinicopathologic Features ◽  
Akt Signaling Pathway ◽  
Potential Therapeutic Target ◽  
Reporter Assays ◽  
The Relationship ◽  
Nucleolar Rna

Background/Aims: Long noncoding RNAs (lncRNAs) have recently emerged as novel and potentially promising therapeutic targets in various cancers. However, the expression pattern and biological function of lncRNAs in glioma remain largely elusive. In the present study, we investigated the functional role of an lncRNA, small nucleolar RNA host gene 16 (SNHG16), in glioma. Methods: The expression levels of SNHG16 and miR-4518 were measured using qRT-PCR. The relationship between the levels of SNHG16 and clinicopathologic features were statically analyzed. The levels of proteins were detected using western blot. Bioinformatics analysis and luciferase reporter assays were applied to the analysis of the relationship between SNHG16, miR-4518 and PRMT5. Cell viability and apoptosis were measured using MTT and apoptosis ELISA assay, respectively. Results: SNHG16 was highly expressed in glioma tissues and cell lines, which was related to poorer clinicopathologic features and shorter survival time. Knockdown of SNHG16 inhibits the viability and induces apoptosis of glioma cells. Further investigation revealed that SNHG16 could up-regulate the expression of miR-4518 targeted gene PRMT5 via acting as an endogenous sponge of miR-4518. Moreover, SNHG16 also affects the expression of Bcl-2 family proteins and the activation of PI3K/Akt signaling pathway. Conclusion: Our study revealed a novel SNHG16-miR-4518-PRMT5 pathway regulatory axis in glioma pathogenesis. SNHG16 could be used as a potential therapeutic target in the treatment of glioma.

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 Rspo3 regulates the abnormal differentiation of small intestinal epithelial cells in diabetic state

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ti-Dong Shan ◽  
Han Yue ◽  
Xue-Guo Sun ◽  
Yue-Ping Jiang ◽  
Li Chen
Keyword(s):  
Epithelial Cells ◽  
Bioinformatics Analysis ◽  
Intestinal Epithelial Cells ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Intestinal Epithelial ◽  
Epithelial Stem Cells ◽  
Diabetic State ◽  
Definitive Evidence

Abstract Background The complications caused by diabetes mellitus (DM) are the focus of clinical treatment. However, little is known about diabetic enteropathy (DE) and its potential underlying mechanism. Methods Intestinal epithelial cells (IECs) and intestinal epithelial stem cells (IESCs) were harvested from BKS.Cg-Dock7m+/+Leprdb/JNju (DM) mice, and the expression of R-Spondin 3 (Rspo3) was detected by RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence. The role of Rspo3 in the abnormal differentiation of IECs during DM was confirmed by knockdown experiments. Through miRNA expression profiling, bioinformatics analysis, and RT-qPCR, we further analyzed the differentiation-related miRNAs in the IECs from mice with DM. Results Abnormal differentiation of IECs was observed in the mice with DM. The expression of Rspo3 was upregulated in the IECs from the mice with DM. This phenomenon was associated with Rspo3 overexpression. Additionally, Rspo3 is a major determinant of Lgr5+ stem cell identity in the diabetic state. Microarray analysis, bioinformatics analysis, and luciferase reporter assays revealed that microRNA (miR)-380-5p directly targeted Rspo3. Moreover, miR-380-5p upregulation was observed to attenuate the abnormal differentiation of IECs by regulating Rspo3 expression. Conclusions Together, our results provide definitive evidence of the essential role of Rspo3 in the differentiation of IECs in DM.

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 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.

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