RÉSUMÉ
Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)-an mRNA-binding protein-is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR. Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.
Sujet(s)
Protéine-1 similaire à ELAV , Naphtoquinones , Facteur de croissance endothéliale vasculaire de type A , Humains , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Protéine-1 similaire à ELAV/antagonistes et inhibiteurs , Facteur de croissance endothéliale vasculaire de type A/métabolisme , Facteur de croissance endothéliale vasculaire de type A/génétique , Facteur de croissance endothéliale vasculaire de type A/antagonistes et inhibiteurs , Naphtoquinones/pharmacologie , Animaux , Souris , Lignée cellulaire tumorale , Souris nude , Gliome/métabolisme , Gliome/traitement médicamenteux , Gliome/anatomopathologie , Tumeurs du cerveau/traitement médicamenteux , Tumeurs du cerveau/métabolisme , Tumeurs du cerveau/anatomopathologie , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Néovascularisation pathologique/traitement médicamenteux , Néovascularisation pathologique/métabolisme , Souris de lignée BALB C , Tests d'activité antitumorale sur modèle de xénogreffe/méthodesRÉSUMÉ
Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.
Sujet(s)
Tumeurs du sein , Protéine-1 similaire à ELAV , Glutaminase , Glutaminase/métabolisme , Glutaminase/génétique , Glutaminase/antagonistes et inhibiteurs , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Humains , Tumeurs du sein/génétique , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Lignée cellulaire tumorale , Femelle , ARN messager/métabolisme , ARN messager/génétique , Régulation de l'expression des gènes tumoraux , Épissage alternatif , Prolifération cellulaire , Glutamine/métabolisme , Stabilité de l'ARNRÉSUMÉ
BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated. METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22's influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status. RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC. CONCLUSION: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.
Sujet(s)
Autophagie , Mouvement cellulaire , Prolifération cellulaire , Sous-unité alpha 2 du facteur CBF , Protéine-1 similaire à ELAV , microARN , ARN circulaire , Protéine FUS de liaison à l'ARN , Tumeurs de l'estomac , p21-Activated Kinases , Humains , Tumeurs de l'estomac/génétique , Tumeurs de l'estomac/anatomopathologie , Tumeurs de l'estomac/métabolisme , ARN circulaire/génétique , ARN circulaire/métabolisme , Autophagie/génétique , microARN/génétique , microARN/métabolisme , p21-Activated Kinases/métabolisme , p21-Activated Kinases/génétique , Prolifération cellulaire/génétique , Protéine FUS de liaison à l'ARN/métabolisme , Protéine FUS de liaison à l'ARN/génétique , Mouvement cellulaire/génétique , Lignée cellulaire tumorale , Animaux , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Sous-unité alpha 2 du facteur CBF/métabolisme , Sous-unité alpha 2 du facteur CBF/génétique , Régulation de l'expression des gènes tumoraux , Souris nude , Souris , Invasion tumorale , Souris de lignée BALB CRÉSUMÉ
BACKGROUND: Endometrial cancer (EC) is the sixth most frequent cancer in women worldwide and has higher fatality rates. The pathophysiology of EC is complex, and there are currently no reliable methods for diagnosing and treating the condition. Long non-coding RNA (lncRNA), according to mounting evidence, is vital to the pathophysiology of EC. HOTAIR is regarded as a significant prognostic indicator of EC. ZBTB7A decreased EC proliferation and migration, according to recent studies, however the underlying mechanism still needs to be clarified. METHODS: The research utilized RT-qPCR to measure HOTAIR expression in clinical EC tissues and various EC cell lines. Kaplan-Meier survival analysis was employed to correlate HOTAIR levels with patient prognosis. Additionally, the study examined the interaction between ZBTB7A and HOTAIR using bioinformatics tools and ChIP assays. The experimental approach also involved manipulating the expression levels of HOTAIR and ZBTB7A in EC cell lines and assessing the impact on various cellular processes and gene expression. RESULTS: The study found significantly higher levels of HOTAIR in EC tissues compared to adjacent normal tissues, with high HOTAIR expression correlating with poorer survival rates and advanced cancer characteristics. EC cell lines like HEC-1 A and KLE showed higher HOTAIR levels compared to normal cells. Knockdown of HOTAIR in these cell lines reduced proliferation, angiogenesis, and migration. ZBTB7A was found to be inversely correlated with HOTAIR, and its overexpression led to a decrease in HOTAIR levels and a reduction in malignant cell behaviors. The study also uncovered that HOTAIR interacts with ELAVL1 to regulate SOX17, which in turn activates the Wnt/ß-catenin pathway, promoting malignant behaviors in EC cells. CONCLUSION: HOTAIR is a critical regulator in EC, contributing to tumor growth and poor prognosis. Its interaction with ZBTB7A and regulation of SOX17 via the Wnt/ß-catenin pathway underlines its potential as a therapeutic target.
Sujet(s)
Prolifération cellulaire , Protéine-1 similaire à ELAV , Tumeurs de l'endomètre , ARN long non codant , Facteurs de transcription SOX-F , Humains , ARN long non codant/génétique , Femelle , Tumeurs de l'endomètre/génétique , Tumeurs de l'endomètre/anatomopathologie , Tumeurs de l'endomètre/métabolisme , Facteurs de transcription SOX-F/génétique , Facteurs de transcription SOX-F/métabolisme , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Néovascularisation pathologique/génétique , Néovascularisation pathologique/anatomopathologie , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Pronostic , Lignée cellulaire tumorale , Régulation de l'expression des gènes tumoraux , Mouvement cellulaire/génétique , Animaux , Souris , Adulte d'âge moyen , Voie de signalisation Wnt/génétique ,RÉSUMÉ
Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.
Sujet(s)
Cytoplasme , Protéine-1 similaire à ELAV , Multimérisation de protéines , Humains , Cytoplasme/métabolisme , Phosphorylation , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Cellules HeLa , Noyau de la cellule/métabolismeRÉSUMÉ
A subarachnoid hemorrhage (SAH) is life-threatening bleeding into the subarachnoid space that causes brain damage. Growing evidence has suggested that melatonin provides neuroprotection following SAH. Exploring the mechanisms underlying melatonin-mediated neuroprotection contributes to its clinical application in SAH. The plasma and cerebrospinal fluid (CSF) were collected from SAH patients, and SAH mice were established via pre-chiasmatic injection. Circodz3 expression, levels of IL-1ß and TNF-α, brain water content, neurological and beam-waling scores were determined. Ferroptosis was evaluated by analyzing levels of iron, lipid ROS, MDA, and GSH. The colocalization of circodz3 and Iba-1 was analyzed by immunofluorescence staining. Interaction of circodz3 and HuR was determined with RNA pull-down and RNA immunoprecipitation assays. Herein, we found that circodz3 was highly abundant in SAH patients and mice. Colocalization of circodz3 and Iba-1 in the left hemisphere of SAH mice suggested the implication of circodz3 in regulating microglia activation following SAH. Melatonin alleviated brain edema, neurological impairment, and microglia activation and inhibited circodz3 expression in SAH mice. Moreover, melatonin inhibited M1 polarization, oxidative stress and ferroptosis and restrained circodz3 expression in primary microglia following SAH. These effects were abrogated by circodz3 overexpression. Circodz3 knockdown inhibited ferroptosis and M1 polarization of BV2 microglia after SAH. Circodz3 interacted with HuR to facilitate ß-Trcp1-mediated ubiquitination and degradation, thus restraining the expression of SLC7A11 and GPX4. Collectively, melatonin exerted neuroprotection following SAH via inhibiting ferroptosis and M1 polarization through the circodz3/HuR axis. Our study suggests potential application of melatonin in the treatment of SAH.
Sujet(s)
Protéine-1 similaire à ELAV , Ferroptose , Mélatonine , Souris de lignée C57BL , Microglie , Hémorragie meningée , Hémorragie meningée/métabolisme , Hémorragie meningée/traitement médicamenteux , Ferroptose/effets des médicaments et des substances chimiques , Ferroptose/physiologie , Animaux , Mélatonine/pharmacologie , Mélatonine/usage thérapeutique , Mélatonine/métabolisme , Microglie/métabolisme , Microglie/effets des médicaments et des substances chimiques , Souris , Humains , Mâle , Protéine-1 similaire à ELAV/métabolisme , ARN circulaire/métabolisme , Neuroprotecteurs/pharmacologie , Neuroprotecteurs/usage thérapeutique , Adulte d'âge moyenRÉSUMÉ
Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.
Sujet(s)
Cytoplasme , Protéine-1 similaire à ELAV , Inflammation , Poly (ADP-Ribose) polymerase-1 , ARN messager , p38 Mitogen-Activated Protein Kinases , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , p38 Mitogen-Activated Protein Kinases/métabolisme , p38 Mitogen-Activated Protein Kinases/génétique , Humains , Poly (ADP-Ribose) polymerase-1/métabolisme , Poly (ADP-Ribose) polymerase-1/génétique , Cytoplasme/métabolisme , Inflammation/métabolisme , Inflammation/génétique , Inflammation/anatomopathologie , ARN messager/métabolisme , ARN messager/génétique , Phosphorylation , Régulation de l'expression des gènes , Animaux , Poly(ADP-ribosylation)/génétique , Cellules HEK293 , Noyau de la cellule/métabolisme , SourisRÉSUMÉ
Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the underlying mechanisms remain to be further studied. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3' UTR of Dgat2 mRNA and intron 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3' UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.
Sujet(s)
Alimentation riche en graisse , Protéine-1 similaire à ELAV , Absorption intestinale , Triglycéride , Triglycéride/métabolisme , Triglycéride/biosynthèse , Animaux , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Souris , Alimentation riche en graisse/effets indésirables , Humains , Souris de lignée C57BL , Mâle , Diacylglycerol O-acyltransferase/métabolisme , Diacylglycerol O-acyltransferase/génétique , Stéatose hépatique non alcoolique/métabolisme , Stéatose hépatique non alcoolique/anatomopathologie , Stéatose hépatique non alcoolique/génétique , Obésité/métabolisme , Obésité/génétique , ARN messager/métabolisme , ARN messager/génétique , Matières grasses alimentaires/métabolisme , Matières grasses alimentaires/pharmacologie , Souris knockout , Régions 3' non traduites/génétique , AcyltransferasesRÉSUMÉ
Circular RNAs (circRNAs) have been discovered to serve as vital regulators in atherosclerosis (AS). However, the role and mechanism of circ_0002331 in AS process are still unclear. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL to establish an in vitro model for AS. The expression levels of circ_0002331, Cyclin D2 (CCND2) and ELAVL1 were analyzed by quantitative real-time PCR. Cell proliferation, apoptosis, migration, invasion and angiogenesis were assessed by EdU assay, flow cytometry, transwell assay and tube formation assay. The protein levels of CCND2, ELAVL1, and autophagy-related markers were detected using western blot analysis. IL-8 level was analyzed by ELISA. The relationship between ELAVL1 and circ_0002331 or CCND2 was analyzed by RIP assay and RNA pull-down assay. Moreover, FISH assay was used to analyze the co-localization of ELAVL1 and CCND2 in HUVECs. Our data showed that circ_0002331 was obviously downregulated in AS patients and ox-LDL-induced HUVECs. Overexpression of circ_0002331 could promote proliferation, migration, invasion and angiogenesis, while inhibit apoptosis, autophagy and inflammation in ox-LDL-induced HUVECs. Furthermore, CCND2 was positively regulated by circ_0002331, and circ_0002331 could bind with ELAVL1 to promote CCND2 mRNA stability. Besides, CCND2 overexpression suppressed ox-LDL-induced HUVECs dysfunction, and its knockdown also reversed the regulation of circ_0002331 on ox-LDL-induced HUVECs dysfunction. In conclusion, circ_0002331 might be a potential target for AS treatment, which could improve ox-LDL-induced dysfunction of HUVECs via regulating CCND2 by binding with ELAVL1.
Sujet(s)
Apoptose , Athérosclérose , Mouvement cellulaire , Prolifération cellulaire , Cycline D2 , Protéine-1 similaire à ELAV , Cellules endothéliales de la veine ombilicale humaine , Lipoprotéines LDL , Stabilité de l'ARN , ARN circulaire , ARN messager , Transduction du signal , Humains , ARN circulaire/métabolisme , ARN circulaire/génétique , Lipoprotéines LDL/toxicité , Lipoprotéines LDL/métabolisme , Cellules endothéliales de la veine ombilicale humaine/métabolisme , Cellules endothéliales de la veine ombilicale humaine/anatomopathologie , Cellules endothéliales de la veine ombilicale humaine/effets des médicaments et des substances chimiques , Cycline D2/métabolisme , Cycline D2/génétique , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Cellules cultivées , Athérosclérose/métabolisme , Athérosclérose/anatomopathologie , Athérosclérose/génétique , ARN messager/métabolisme , ARN messager/génétique , Régulation de l'expression des gènes , Études cas-témoins , Autophagie , Mâle , Adulte d'âge moyen , FemelleRÉSUMÉ
MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3' untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3'UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3'UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3'UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.
Sujet(s)
Régions 3' non traduites , Carcinome hépatocellulaire , Protéine-1 similaire à ELAV , Régulation de l'expression des gènes tumoraux , Tumeurs du foie , MAP Kinase Kinase Kinase 2 , microARN , Humains , microARN/génétique , microARN/métabolisme , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/métabolisme , Carcinome hépatocellulaire/anatomopathologie , Tumeurs du foie/génétique , Tumeurs du foie/métabolisme , Tumeurs du foie/anatomopathologie , Régions 3' non traduites/génétique , MAP Kinase Kinase Kinase 2/métabolisme , MAP Kinase Kinase Kinase 2/génétique , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Lignée cellulaire tumorale , Biosynthèse des protéinesRÉSUMÉ
Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients. Pharmacological inhibition of TRPM7 protected primary RA-FLSs from proliferation, metastasis and inflammation. Furthermore, we found that TRPM7 contributes to RA-FLS proliferation, metastasis and inflammation by increasing the intracellular Ca2+ concentration. Mechanistically, the PKCα-HuR axis was demonstrated to respond to Ca2+ influx, leading to TRPM7-mediated RA-FLS proliferation, metastasis and inflammation. Moreover, HuR was shown to bind to IL-6 mRNA after nuclear translocation, which could be weakened by TRPM7 channel inhibition. Additionally, adeno-associated virus 9-mediated TRPM7 silencing is highly effective at alleviating synovial hyperplasia and inflammation in adjuvant-induced arthritis rats. In conclusion, our findings unveil a novel regulatory mechanism involved in the pathogenesis of RA and suggest that targeting TRPM7 might be a potential strategy for the prevention and treatment of RA.
Sujet(s)
Arthrite expérimentale , Polyarthrite rhumatoïde , Prolifération cellulaire , Interleukine-6 , Protein kinase C-alpha , Cellules synoviales , Canaux cationiques TRPM , Canaux cationiques TRPM/métabolisme , Canaux cationiques TRPM/génétique , Polyarthrite rhumatoïde/anatomopathologie , Polyarthrite rhumatoïde/métabolisme , Animaux , Cellules synoviales/métabolisme , Cellules synoviales/anatomopathologie , Humains , Interleukine-6/métabolisme , Interleukine-6/génétique , Protein kinase C-alpha/métabolisme , Protein kinase C-alpha/génétique , Arthrite expérimentale/anatomopathologie , Arthrite expérimentale/métabolisme , Mâle , Rats , Fibroblastes/métabolisme , Fibroblastes/anatomopathologie , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Protein-Serine-Threonine Kinases/métabolisme , Protein-Serine-Threonine Kinases/génétique , Cellules cultivées , Inflammation/métabolisme , Inflammation/anatomopathologie , Rat Sprague-Dawley , Femelle , Transduction du signalRÉSUMÉ
AIMS/INTRODUCTION: Tanshinone IIA (TIIA) is one of the main components of the root of the red-rooted Salvia miltiorrhiza Bunge. However, the molecular mechanisms underlying TIIA-mediated protective effects in diabetic nephropathy (DN) are still unclear. MATERIALS AND METHODS: High glucose (HG)-induced mouse podocyte cell line (MPC5) cells were used as the in vitro model of DN and treated with TIIA. Cell viability, proliferation and apoptosis were detected using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine and flow cytometry assays. The protein levels were assessed using western blot assay. The levels of inflammatory factors were deleted by enzyme-linked immunoassay. Fe+ level, reactive oxygen species, malondialdehyde and glutathione products were detected using special assay kits. After ENCORI prediction, the interaction between embryonic lethal abnormal visual-like protein 1 (ELAVL1) and acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) was verified using co-immunoprecipitation assay and dual-luciferase reporter assays. ACSL4 messenger ribonucleic acid expression was measured using real-time quantitative polymerase chain reaction. RESULTS: TIIA repressed HG-induced MPC5 cell apoptosis, inflammatory response and ferroptosis. ACSL4 upregulation relieved the repression of TIIA on HG-mediated MPC5 cell injury and ferroptosis. ELAVL1 is bound with ACSL4 to positively regulate the stability of ACSL4 messenger ribonucleic acid. TIIA hindered HG-triggered MPC5 cell injury and ferroptosis by regulating the ELAVL1-ACSL4 pathway. TIIA blocked DN progression in in vivo research. CONCLUSION: TIIA treatment restrained HG-caused MPC5 cell injury and ferroptosis partly through targeting the ELAVL1-ACSL4 axis, providing a promising therapeutic target for DN treatment.
Sujet(s)
Abiétanes , Coenzyme A ligases , Néphropathies diabétiques , Ferroptose , Podocytes , Transduction du signal , Abiétanes/pharmacologie , Ferroptose/effets des médicaments et des substances chimiques , Animaux , Podocytes/effets des médicaments et des substances chimiques , Podocytes/métabolisme , Podocytes/anatomopathologie , Néphropathies diabétiques/métabolisme , Néphropathies diabétiques/traitement médicamenteux , Néphropathies diabétiques/anatomopathologie , Souris , Transduction du signal/effets des médicaments et des substances chimiques , Coenzyme A ligases/métabolisme , Protéine-1 similaire à ELAV/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Lignée cellulaireRÉSUMÉ
RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.
Sujet(s)
Liaison aux protéines , Protéines de liaison à l'ARN , ARN , Humains , ARN/métabolisme , ARN/génétique , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Immunoprécipitation/méthodes , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Noyau de la cellule/métabolisme , Noyau de la cellule/génétique , Granulations cytoplasmiques/métabolisme , Arsénites , Cellules HeLa , Cytosol/métabolisme , Cellules HEK293RÉSUMÉ
Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1ß-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1ß-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1ß induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1ß-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1ß-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1ß-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1ß. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1ß-induced IL-8 production through this non-transcriptional mechanism.
Sujet(s)
Noyau de la cellule , Cytoplasme , Protéine-1 similaire à ELAV , Interleukine-1 bêta , Interleukine-8 , Facteur de transcription STAT-3 , Humains , Facteur de transcription STAT-3/métabolisme , Facteur de transcription STAT-3/génétique , Interleukine-1 bêta/métabolisme , Interleukine-8/métabolisme , Interleukine-8/génétique , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Cytoplasme/métabolisme , Noyau de la cellule/métabolisme , Lignée cellulaire tumorale , S-Oxydes cycliques/pharmacologie , Transport des protéines , Transduction du signal , Transport nucléaire actif , Systèmes CRISPR-CasRÉSUMÉ
BACKGROUND AND AIMS: Long noncoding RNAs (lncRNAs) play important roles in the progression of atherosclerosis. In this study, we identified an uncharacterized lncRNA, Liver Expressions by PSRC1 Induced Specifically (LEPIS). This study aimed to clarify the mechanism though which LEPIS affects atherosclerosis (AS). METHODS: The expression of LEPIS and its potential target, tropomodulin 4 (TMOD4), was increased in the livers of ApoE-/- mice fed a high-fat diet (HFD). An ApoE-/- mouse model in which LEPIS or TMOD4 was overexpressed in the liver was established. The plaque load in the aorta was assessed, plasma was collected to measure blood lipid levels, and the liver was collected to study cholesterol metabolism. RESULTS: We found that both LEPIS and TMOD4 increased the AS burden and reduced hepatic cholesterol levels. A further study revealed that LEPIS and TMOD4 affected the expression of genes related to hepatic cholesterol homeostasis, including proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR), which are closely related to hypercholesterolemia. Mechanistically, human antigen R (HuR), an RNA-binding protein (RBP), was shown to be critical for the regulation of TMOD4 by LEPIS. Furthermore, we found that verexpression of LEPIS promoted the shuttling of HuR from the nucleus to the cytoplasm, enhanced the stability of TMOD4 mRNA, and in turn promoted the expression of TMOD4. In addition, TMOD4 was found to affect intracellular cholesterol levels through PCSK9. CONCLUSIONS: These results suggest that the LEPIS-HuR-TMOD4 axis is a potential intervention target for dysregulated hepatic cholesterol homeostasis and AS and may provide the basis for further reductions in the circulating LDL-C concentration and arterial plaque burden.
Sujet(s)
Athérosclérose , Cholestérol , Modèles animaux de maladie humaine , Homéostasie , Foie , Souris invalidées pour les gènes ApoE , Animaux , Humains , Mâle , Souris , Maladies de l'aorte/métabolisme , Maladies de l'aorte/génétique , Maladies de l'aorte/anatomopathologie , Athérosclérose/métabolisme , Athérosclérose/génétique , Athérosclérose/anatomopathologie , Cholestérol/métabolisme , Cholestérol/sang , Alimentation riche en graisse , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Foie/métabolisme , Souris de lignée C57BL , Plaque d'athérosclérose , Proprotéine convertase 9/métabolisme , Proprotéine convertase 9/génétique , Récepteurs aux lipoprotéines LDL/génétique , Récepteurs aux lipoprotéines LDL/métabolisme , ARN long non codant/génétique , ARN long non codant/métabolismeRÉSUMÉ
The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.
Sujet(s)
Carcinome hépatocellulaire , Tumeurs du foie , Animaux , Humains , Souris , Carcinome hépatocellulaire/métabolisme , Modèles animaux de maladie humaine , Protéine-1 similaire à ELAV/métabolisme , Tumeurs du foie/anatomopathologie , ARN/métabolisme , SumoylationRÉSUMÉ
Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.
Sujet(s)
Protéine-1 similaire à ELAV , Macrophages , microARN , Protéines Qa-SNARE , Animaux , Humains , Souris , Endosomes/métabolisme , Leishmania donovani/métabolisme , Leishmania donovani/génétique , Activation des macrophages , Macrophages/métabolisme , microARN/métabolisme , microARN/génétique , Protéines Qa-SNARE/métabolisme , Protéines Qa-SNARE/génétique , Transport des ARN , Protéine-1 similaire à ELAV/métabolismeRÉSUMÉ
RNA-binding proteins (RBPs) play a crucial role in the regulation of posttranscriptional RNA networks, which can undergo dysregulation in many pathological conditions. Human antigen R (HuR) is a highly researched RBP that plays a crucial role as a posttranscriptional regulator. HuR plays a crucial role in the amplification of inflammatory signals by stabilizing the messenger RNA of diverse inflammatory mediators and key molecular players. The noteworthy correlations between HuR and its target molecules, coupled with the remarkable impacts reported on the pathogenesis and advancement of multiple diseases, position HuR as a promising candidate for therapeutic intervention in diverse inflammatory conditions. This review article examines the significance of HuR as a member of the RBP family, its regulatory mechanisms, and its implications in the pathophysiology of inflammation and cardiometabolic illnesses. Our objective is to illuminate potential directions for future research and drug development by conducting a comprehensive analysis of the existing body of research on HuR.
Sujet(s)
Maladies cardiovasculaires , Protéine-1 similaire à ELAV , Inflammation , Humains , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Inflammation/génétique , Inflammation/anatomopathologie , Maladies cardiovasculaires/génétique , Maladies cardiovasculaires/immunologie , Maladies cardiovasculaires/métabolisme , Animaux , Régulation de l'expression des gènes , Maladies métaboliques/génétique , Maladies métaboliques/immunologie , Maladies métaboliques/métabolisme , Transduction du signal , Protéines de liaison à l'ARN/génétique , Protéines de liaison à l'ARN/métabolismeRÉSUMÉ
There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.
Sujet(s)
Carcinome hépatocellulaire , Cycline D1 , Protéine-1 similaire à ELAV , Régulation de l'expression des gènes tumoraux , Tumeurs du foie , ARN long non codant , Transduction du signal , Animaux , Humains , Souris , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/métabolisme , Carcinome hépatocellulaire/anatomopathologie , Carcinome hépatocellulaire/virologie , Lignée cellulaire tumorale , Prolifération cellulaire , Cycline D1/métabolisme , Cycline D1/génétique , Protéine-1 similaire à ELAV/métabolisme , Protéine-1 similaire à ELAV/génétique , Tumeurs du foie/génétique , Tumeurs du foie/métabolisme , Tumeurs du foie/anatomopathologie , Tumeurs du foie/virologie , Souris nude , ARN long non codant/génétique , ARN long non codant/métabolisme , Transduction du signal/génétique , Hépatite C/complications , Régulation positive , Marqueurs biologiques tumorauxRÉSUMÉ
Breast cancer (BC) is the most prevalent malignant tumor in women worldwide with high morbidity and mortality. NSUN2, a crucial RNA methyltransferase, plays a pivotal role in regulating the proliferation and metastasis of tumor cells. Our study demonstrated that NSUN2 is upregulated in BC tissues and cell lines, and its high expression is associated with a poor prognosis in BC patients. Knockout of NSUN2 exerted inhibitory effects on the proliferation and migration of BC cells in vitro and in vivo. Mechanistic investigations revealed that the RNA-binding protein ELAVL1 can bind to NSUN2 mRNA and increase its stability. Additionally, we identified HOST2, a long non-coding RNA, as a key player in blocking the ubiquitin-dependent proteasomal degradation of ELAVL1, thereby influencing the stability of NSUN2 mRNA. In conclusion, this study revealed for the first time that HOST2 maintains NSUN2 mRNA stability by blocking ubiquitin-dependent degradation of ELAVL1, which in turn affects BC progression. HOST2/ELAVL1/NSUN2 oncogenic cascade has the potential to be a novel therapeutic target for BC.