The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-rich-element-dependent manner.
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ABSTRACT:
SUBMITTER: Dormoy-Raclet V
PROVIDER: S-EPMC1952093 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.
| S-EPMC139819 | biostudies-literature
Project description:The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3'-untranslated region. When this 217-nucleotide nutrient sensor AU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.
| S-EPMC1959406 | biostudies-literature
Project description:Stromal interaction molecule 1 (Stim1) functions as a sensor of Ca2+ within stores and plays an essential role in the activation of store-operated Ca2+ entry (SOCE). Although lowering Stim1 levels reduces store-operated Ca2+ entry and inhibits intestinal epithelial repair after wounding, the mechanisms that control Stim1 expression remain unknown. Here, we show that cellular Stim1 abundance is controlled posttranscriptionally via factors that associate with 3'-untranslated region (3'-UTR) of stim1 mRNA. MicroRNA-195 (miR-195) and the RNA-binding protein HuR competed for association with the stim1 3'-UTR and regulated stim1 mRNA decay in opposite directions. Interaction of miR-195 with the stim1 3'-UTR destabilized stim1 mRNA, whereas the stability of stim1 mRNA increased with HuR association. Interestingly, ectopic miR-195 overexpression enhanced stim1 mRNA association with argonaute-containing complexes and increased the colocalization of tagged stim1 RNA with processing bodies (P-bodies); the translocation of stim1 mRNA was abolished by HuR overexpression. Moreover, decreased levels of Stim1 by miR-195 overexpression inhibited cell migration over the denuded area after wounding but was rescued by increasing HuR levels. In sum, Stim1 expression is controlled by two factors competing for influence on stim1 mRNA stability: the mRNA-stabilizing protein HuR and the decay-promoting miR-195.
| S-EPMC3763549 | biostudies-literature
Project description:Bone metastasis is the major cause of morbidity and mortality in patients with prostate cancer (PCa). However, the underlying mechanism of bone-specific metastasis remain vague. Recently, with the deep research of N6-methyladenine (m6A) mRNA methylation, many studies directly focus on the role of m6A modification in human diseases, especially in cancers. Here we found that methyltransferase-like 3 (METTL3) expression is higher in PCa than in normal prostate tissues, especially in PCa with bone metastasis. High METTL3 expression was positively correlated with advanced progression and a poor prognosis of PCa. Functional assays demonstrated that METTL3 regulates the expression of Integrin β1 (ITGB1) through m6A-HuR-dependent mechanism, which affects the binding of ITGB1 to Collagen I and tumor cell motility, so as to promote the bone metastasis of PCa. The findings of this study reveal a novel mechanism of PCa osteotropism and suggest METTL3 as a therapeutic target for PCa bone metastasis.
| S-EPMC7136910 | biostudies-literature
Project description:PurposeIn an earlier study, we showed that human antigen R (HuR) and β-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects β-actin gene expression and in turn affects corneal fibroblast migration and wound healing.MethodsStromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for β-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively.ResultsReverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, β-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the β-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced.ConclusionsThe results show that downregulation of the HuR gene results in decreased β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased β-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.
| S-EPMC4016806 | biostudies-literature
Project description:Circular RNAs (circRNAs) have been implicated in tumorigenesis and progression of various cancers. However, the underlying mechanisms of circRNAs in hepatocellular carcinoma (HCC) have not been fully elucidated. Herein, a new oncogenic circRNA, hsa_circ_0070039 (circNUP54), was identified to be significantly upregulated in HCC through circRNA sequencing. As verified in 68 HCC samples, circNUP54 overexpression was correlated with aggressive cancerous behaviors and poor outcomes. Moreover, the function experiments showed that knockdown of circNUP54 inhibited the malignant progression of HCC in vitro and in vivo, whereas overexpression of circNUP54 had the opposite role. Mechanistic investigations carried out by RNA pull-down, RNA immunoprecipitation, and immunofluorescence revealed that circNUP54 interacted with the RNA-binding protein Hu-antigen R (HuR) and promoted its cytoplasmic export. The cytoplasmic accumulation of HuR stabilized the downstream BIRC3 mRNA through its binding to the 3' UTR region. Consequently, the encoded protein of BIRC3, cellular inhibitor of apoptosis 2 (cIAP2), proceeded to activate the NF-κB signal pathway and ultimately contributed to HCC progression. In addition, depletion of BIRC3 rescued the pro-tumorigenic effect of circNUP54 on HCC cells. Overall, this study demonstrated that circNUP54 facilitates HCC progression via regulating the HuR/BIRC3/NF-κB axis, which may serve as a promising therapeutic target for HCC treatment.
| S-EPMC10914787 | biostudies-literature
Project description:Triple-negative breast cancer (TNBC) is a molecular subtype with an unfavorable prognosis, and metastasis is the main reason for the failure of clinical treatment. However, the expression profile and regulatory function of circRNAs in TNBC progression are not fully understood. Herein, we performed high-throughput RNA-seq in paired breast cancer tissues and adjacent normal tissues and discovered a novel circRNA, circEIF3H, which was upregulated in breast cancer tissues. Large cohort survival analysis confirmed the association between high circEIF3H expression and poor prognosis of TNBC, indicating the vital function of circEIF3H in TNBC progression. Then we conducted both in vitro and in vivo experiments which illustrated that circEIF3H was essential for TNBC proliferation and metastasis. Further experiments showed that circEIF3H did not function as a microRNA sponge as in the most well-established pathway, but as a scaffold for IGF2BP2 and HuR to regulate the mRNA stability of HSPD1, RBM8A, and G3BP1. Our findings provide insight into a novel circRNA, circEIF3H, with significant cancer-promoting function via serving as a scaffold for IGF2BP2/HuR. These results identified circEIF3H as a potential target for developing individualized therapy of TNBC in the approaching future.
| S-EPMC9107465 | biostudies-literature
Project description:Our previous study shows that cellular retinoic acid binding protein II (CRABP-II) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and pre-cancerous lesions, but not detected in normal pancreatic tissues. In this study, we show that deletion of CRABP-II in PDAC cells by CRISPR/Cas9 does not affect cancer cell proliferation, but decreases cell migration and invasion. Gene expression microarray analysis reveals that IL-8 is one of the top genes whose expression is down-regulated upon CRABP-II deletion, while expression of MMP-2 and MMP-14, two targets of IL-8 are also significantly down-regulated. Moreover, we found that CRABP-II is able to form a complex with HuR, which binds to the 3'UTR of IL-8 messenger RNA (mRNA) and enhances IL-8 mRNA stability. Ectopic expression of flag-CRABP-II in CRABP-II knockout cells is able to rescue the expression of IL-8, MMP-2/MMP-14 and recovers cell migration. Using the orthotopic xenograft model, we further demonstrate that CRABP-II deletion impairs tumor metastasis to nearby lymph nodes. Taken together, our results reveal a novel pathway linking CRABP-II expression to enhanced PDAC metastasis, and hence we propose CRABP-II may serve as a new PDAC therapeutic target.
| S-EPMC5581040 | biostudies-literature
Project description:BackgroundHypoxic microenvironments play a significant role in the progression of colorectal cancer (CRC). Silencing information regulator 1 (SIRT1), a class III histone deacetylase, modulates the multiple biological behaviors of cancer. However, its role in CRC remains unclear. This study aims to explore the role of SIRT1 in CRC migration and invasion under hypoxia.MethodsSIRT1 protein and mRNA levels were detected by Western blotting and real-time PCR in CRC cells exposed to hypoxia (1% O2). The migration and invasion abilities of SW480 and HCT116 cells with SIRT1 overexpression or knockdown were studied with transwell assays, and the results were confirmed by those of treatment with specific SIRT1 activator (SRT1720) and inhibitor (EX527). The dual-luciferase reporter systems with a series of SIRT1 promoter truncations were used to analyze their transcriptional activities, respectively. After a bioinformatic analysis of potential transcription factors, the direct interaction between the transcription factor and SIRT1 promoter was determined by chromatin immunoprecipitation (ChIP) assays. Western blot and real-time PCR assays were used to detect the activation and acetylation levels of the NF-κB pathway.ResultsThe protein and mRNA levels of SIRT1 were significantly decreased under hypoxia, and these effects were replicated by cobalt chloride treatment. Hypoxia promoted cell migration and invasion, which were impeded by the overexpression or activation of SIRT1 and promoted by the knockdown or inhibition of SIRT1. The dual-luciferase reporter gene and ChIP analyses revealed that the core regulatory elements located 100 bp upstream of the SIRT1 promoter and early growth response factor 1 (EGR1) could interact with this DNA sequence. Subsequent rescue experiments suggested that EGR1 was essential for hypoxia-mediated SIRT1 transcriptional suppression. Western blot analyses demonstrated that SIRT1 overexpression eliminated the p65 acetylation induced by hypoxia along with the decreased MMP-2/-9, suggesting that NF-κB was a direct downstream target of SIRT1 and might regulate cell migration and invasion through MMP-2/-9.ConclusionsOur results establish for the first time that EGR1 plays an important role in regulating SIRT1 expression under hypoxia. Hypoxia promotes CRC cell migration and invasion in a SIRT1-dependent manner. And a potential SIRT1/NF-κB/MMP-2/-9 axis modulates this process.
| S-EPMC6492435 | biostudies-literature
Project description:Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.
| S-EPMC162218 | biostudies-literature