Human Antigen R Research Articles

HuR facilitates miR-93-5p-induced activation of MAP3K2 translation via MAP3K2 3′UTR ARE2 in hepatocellular carcinoma

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.

Abstract 3053: An Emerging Role Of The RNA Binding Protein Human Antigen R In Atherosclerotic Vascular Smooth Muscle Cell Heterogeneity

Objective: Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Downregulation of VSMC migration and cytokine release in Par2 -/- mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers, Par2 -/- VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in Par2 -/- VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female Ldlr -/- HuR flox/flox SM22 Cre+ mice have attenuated atherosclerosis compared to their littermate Cre - controls at both 12 and 24 weeks. Conclusions: These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.

Noradrenaline enhances Na-K ATPase subunit expression by HuR-induced mRNA stabilization and their transportation to the cell surface through PLC and PKC mediated pathway: Implications with REMS-loss associated disorders.

Rapid eye movement sleep (REMS) maintains brain excitability at least by regulating Na-K ATPase activity. Although REMS deprivation (REMSD)-associated elevated noradrenaline (NA) increases Na-K ATPase protein expression, its mRNA transcription did not increase. We hypothesized and confirmed both invivo as well as invitro that elevated mRNA stability explains the apparent puzzle. The mRNA stability was measured in control and REMSD rat brain with or without invivo treatment with α1-adrenoceptor (AR) antagonist, prazosin (PRZ). Upon REMSD, Na-K ATPase α1-, and α2-mRNA stability increased significantly, which was prevented by PRZ. To decipher the molecular mechanism of action, we estimated NA-induced Na-K ATPase mRNA stability in Neuro-2a cells under controlled conditions and by transcription blockage using Actinomycin D (Act-D). NA increased Na-K ATPase mRNA stability, which was prevented by PRZ and propranolol (PRP, β-AR antagonist). The knockdown assay confirmed that the increased mRNA stabilization was induced by elevated cytoplasmic abundance of Human antigen R (HuR) and involving (Phospholipase C) PLC-mediated activation of Protein Kinase C (PKC). Additionally, using cell-impermeable Enz-link sulfo NHS-SS-Biotin, we observed that NA increased Na-K ATPase α1-subunits on the Neuro-2a cell surface. We conclude that REMSD-associated elevated NA, acting on α1- and β-AR, increases nucleocytoplasmic translocation of HuR and increases Na-K ATPase mRNA stability, resulting in increased Na-K ATPase protein expression. The latter then gets translocated to the neuronal membrane surface involving both PKC and (Protein Kinase A) PKA-mediated pathways. These findings may be exploited for the amelioration of REMSD-associated chronic disorders and symptoms.

Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

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.

Unravelling driver genes as potential therapeutic targets in ovarian cancer via integrated bioinformatics approach

Target-driven cancer therapy is a notable advancement in precision oncology that has been accompanied by substantial medical accomplishments. Ovarian cancer is a highly frequent neoplasm in women and exhibits significant genomic and clinical heterogeneity. In a previous publication, we presented an extensive bioinformatics study aimed at identifying specific biomarkers associated with ovarian cancer. The findings of the network analysis indicate the presence of a cluster of nine dysregulated hub genes that exhibited significance in the underlying biological processes and contributed to the initiation of ovarian cancer. Here in this research article, we are proceeding our previous research by taking all hub genes into consideration for further analysis. GEPIA2 was used to identify patterns in the expression of critical genes. The KM plotter analysis indicated that the out of all genes 5 genes are statistically significant. The cBioPortal platform was further used to investigate the frequency of genetic mutations across the board and how they affected the survival of the patients. Maximum mutation was reported by ELAVL2. In order to discover viable therapeutic candidates after competitive inhibition of ELAVL2 with small molecular drug complex, high throughput screening and docking studies were used. Five compounds were identified. Overall, our results suggest that the ELAV-like protein 2-ZINC03830554 complex was relatively stable during the molecular dynamic simulation. The five compounds that have been found can also be further examined as potential therapeutic possibilities. The combined findings suggest that ELAVL2, together with their genetic changes, can be investigated in therapeutic interventions for precision oncology, leveraging early diagnostics and target-driven therapy.

Schwann cell-derived extracellular vesicles promote memory impairment associated with chronic neuropathic pain.

The pathogenesis of memory impairment, a common complication of chronic neuropathic pain (CNP), has not been fully elucidated. Schwann cell (SC)-derived extracellular vesicles (EVs) contribute to remote organ injury. Here, we showed that SC-EVs may mediate pathological communication between SCs and hippocampal neurons in the context of CNP. We used an adeno-associated virus harboring the SC-specific promoter Mpz and expressing the CD63-GFP gene to track SC-EVs transport. microRNA (miRNA) expression profiles of EVs and gain-of-function and loss-of-function regulatory experiments revealed that miR-142-5p was the main cargo of SC-EVs. Next, luciferase reporter gene and phenotyping experiments confirmed the direct targets of miR-142-5p. The contents and granule sizes of plasma EVs were significantly greater in rats with chronic sciatic nerve constriction injury (CCI)than in sham rats. Administration of the EV biogenesis inhibitor GW4869 ameliorated memory impairment in CCI rats and reversed CCI-associated dendritic spine damage. Notably, during CCI stress, SC-EVs could be transferred into the brain through the circulation and accumulate in the hippocampal CA1-CA3 regions. miR-142-5p was the main cargo wrapped in SC-EVs and mediated the development of CCI-associated memory impairment. Furthermore, α-actinin-4 (ACTN4), ELAV-like protein 4 (ELAVL4) and ubiquitin-specific peptidase 9 X-linked (USP9X) were demonstrated to be important downstream target genes for miR-142-5p-mediated regulation of dendritic spine damage in hippocampal neurons from CCI rats. Together, these findings suggest that SCs-EVs and/or their cargo miR-142-5p may be potential therapeutic targets for memory impairment associated with CNP.

Role and mechanism of COL3A1 in regulating the growth, metastasis, and drug sensitivity in cisplatin-resistant non-small cell lung cancer cells

ABSTRACT Non-small cell lung cancer (NSCLC) is among the most difficult malignancies to treat. Type III collagen (COL3A1) can affect the progression and chemoresistance development of NSCLC. We herein explored the mechanism that drives COL3A1 dysregulation in NSCLC. Potential RNA-binding proteins (RBPs) and transcription factors (TFs) that could bind to COL3A1 were searched by bioinformatics. mRNA expression was detected by quantitative PCR. Protein expression was evaluated using immunoblotting and immunohistochemistry. The effects of the variables were assessed by gauging cell growth, invasiveness, migratory capacity, apoptosis, and cisplatin (DDP) sensitivity. The direct YY1/COL3A1 relationship was confirmed by ChIP and luciferase reporter experiments. Xenograft experiments were done to examine COL3A1’s function in DDP efficacy. COL3A1 showed enhanced expression in DDP-resistant NSCLC. In H460/DDP and A549/DDP cells, downregulation of COL3A1 exerted inhibitory functions in cell growth, invasiveness, and migration, as well as promoting effects on cell DDP sensitivity and apoptosis. Mechanistically, ELAV-like RNA binding protein 1 (ELAVL1) enhanced the mRNA stability and expression of COL3A1, and Yin Yang 1 (YY1) promoted the transcription and expression of COL3A1. Furthermore, upregulation of COL3A1 reversed ELAVL1 inhibition- or YY1 deficiency-mediated functions in DDP-resistant NSCLC cells. Additionally, COL3A1 downregulation enhanced the anti-tumor efficacy of DDP in vivo. Our investigation demonstrates that COL3A1 upregulation, induced by both RBP ELAVL1 and TF YY1, exerts important functions in phenotypes of NSCLC cells with DDP resistance, offering an innovative opportunity in the treatment of drug-resistant NSCLC.

Abstract 5875: Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance

Abstract Background and Aim: Multidrug resistance (MDR) is an unresolved obstacle to cancer chemotherapy. It is usually caused by overexpression of drug efflux transporters including ABCG2. Exosomes are extracellular lipid vesicles released from all cells upon the fusion of multivesicular bodies with plasma membrane. Tumor-derived exosomes have been shown to mediate cell-to-cell transfer of oncogenic materials to promote drug resistance. Human antigen R (HuR) is an extensively studied RNA binding protein that regulates the stability, translation, and nucleus-to-cytoplasm shuttling of mRNAs. Numerous HuR-targeting mRNAs play crucial roles in tumorigenesis, invasion and metastasis. We have previously reported the induction of ABCG2 and MDR phenotype by HuR overexpression in colon cancer. HuR overexpression is linked with high-grade malignancy and poor prognosis in cancer patients. This study investigated the enrichment of HuR inducing microRNAs (miRNAs) in exosomes secreted from MDR colon cancer and its intracellular transfer to drive MDR. Method: Cancer exosome-mediated transfer of miRNAs from ABCG2-overexpressing resistant colon cancer cells S1M1-80 to a few sensitive colon cell lines was evaluated. Exosomes were isolated from S1M1-80 conditioned medium by differential centrifugation method. The uptake of fluorescence-labeled exosome from donor MDR cancer cells by recipient sensitive cells was visualized by fluorescence microscopy. MiRNA microarray was conducted to identify differentially expressed miRNAs in drug sensitive cells after incubation with S1M1-80-secreted exosomes. Western blot and qPCR analysis were conducted to measure HuR and ABCG2 expression. Result: Exosomes were isolated from resistant S1M1-80 cells, whose purity was confirmed by the presence of exosomal markers (Tsg101 and Alix) but absence of cellular marker (GRP78) by Western blot analysis. These exosomes reduced the apoptotic effect of SN-38 or 5-fluorouracil in sensitive colon cancer cell lines (HCT116, S1, and SW480) by inducing both HuR and ABCG2. A common upregulated miRNA (miR-155-5p) was identified by miRNA microarray in the sensitive cell lines after incubation with S1M1-80-derived exosomes. MiR-155-5p was found to induce HuR expression by promoting its translation. MiR-155-5p inhibitor was shown to reverse the upregulation of HuR in HCT116 cells after exosome incubation, which subsequently suppressed ABCG2 expression and the MDR phenotype. Furthermore, inhibition of exosome uptake by dynasore was found to prevent the induction of HuR/ABCG2, and re-sensitize the exosome-treated sensitive cells to SN38. Conclusion: MDR cells-derived exosomes may spread the drug resistance phenotype by transferring a miR-155-5p/HuR/ABCG2 regulatory machinery to sensitive cells. Inhibition of this exosomal transfer of regulatory materials may prevent the spread of MDR and sensitize cancer cells to chemotherapy. Citation Format: Kenneth K.W. To. Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5875.

Loss of RNA-binding protein CELF2 promotes acute leukemia development via FAT10-mTORC1

RNA-binding proteins (RBPs) are critical regulators for RNA transcription and translation. As a key member of RBPs, ELAV-like family protein 2 (CELF2) has been shown to regulate RNA splicing and embryonic hematopoietic development and was frequently seen dysregulated in acute myeloid leukemia (AML). However, the functional role(s) of CELF2 in hematopoiesis and leukemogenesis has not been fully elucidated. In the current study, we showed that Celf2 deficiency in hematopoietic system led to enhanced HSCs self-renewal and differentiation toward myeloid cells in mice. Loss of Celf2 accelerated myeloid cell transformation and AML development in MLL-AF9-induced AML murine models. Gene expression profiling integrated with RNA immunoprecipitation sequencing (RIP-Seq), together with biochemical experiments revealed that CELF2 deficiency stabilizes FAT10 mRNA, promotes FAT10 translation, thereby increases AKT phosphorylation and mTORC1 signaling pathway activation. Notably, combination therapy with a mTORC1 inhibitor (Rapamycin) and a MA9/DOTL1 inhibitor (EPZ-5676) reduced the leukemia burden in MLL-AF9 mice lacking Celf2 in vivo. Our study elucidated a novel mechanism by which the CELF2/FAT10-AKT/mTORC1 axis regulates the proliferation of normal blood cells and the development of AML, thus providing potential therapeutic targets for myeloid leukemia suppression.

Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages

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 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 Syntaxin5 (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 HuR (Human antigen R). This activity ensures 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.

Human antigen R: Exploring its inflammatory response impact and significance in cardiometabolic disorders.

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 RNAof 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.

LncRNA MEG3 Reduces the Ratio of M2/M1 Macrophages Through the HuR/CCL5 Axis in Hepatocellular Carcinoma.

Tumor-associated macrophages play a crucial role in the development of hepatocellular carcinoma (HCC). Our study aimed to investigate the relationship between long coding RNA (lncRNA) maternally expressed gene 3 (MEG3), RNA-binding protein human antigen R (HuR), and messenger RNA C-C motif chemokine 5 (CCL5) in the modulation of M1 and M2 macrophage polarization in HCC. To induce M1 or M2 polarization, LPS/IFNγ- or IL4/IL13 were used to treat bone marrow derived macrophages (BMDMs). The localization of MEG3 in M1 and M2 macrophages was assessed using fluorescence in situ hybridization assay. Expression levels of MEG3, HuR, CCL5, M1, and M2 markers were measured by RT-qPCR or immunofluorescence staining. Flow cytometry was performed to determine the proportion of F4/80+CD206+ and F4/80+CD68+ cells. RNA pulldown assay was performed to detect the binding of lncRNA MEG3 and HuR. The impacts of HuR on CCL5 stability and activity of CCL5 promoter were evaluated using actinomycin D treatment and luciferase reporter assay. Cell migration, invasiveness, and angiogenesis were assessed using transwell migration and invasion assays and a tube formation assay. A mixture of Huh-7 cells and macrophages were injected into nude mice to explore the effect of MEG3 on tumorigenesis. MEG3 promoted M1-like polarization while dampening M2-like polarization of BMDMs. MEG3 bound to HuR in M1 and M2 macrophages. HuR downregulated CCL5 by inhibiting CCL5 transcription in macrophages. In addition, overexpression of MEG3 suppressed cell metastasis, invasion, and angiogenesis by obstructing macrophage M2 polarization. MEG3 inhibited tumorigenesis in HCC via promotion of M1-like polarization and inhibition of M2-like polarization. Rescue experiments showed that depletion of CCL5 in M2 macrophages reversed MEG3-induced suppressive effect on cell migration, invasion, and tube formation. MEG3 suppresses HCC progression by promoting M1-like while inhibiting M2-like macrophage polarization via binding to HuR and thus upregulating CCL5.

WITHDRAWN: Exploring the Interplay of m6A regulators in renal cell carcinoma: Molecular insights and clinical implications

BackgroundThe m6A modification of N6-methyladenosine (m6A) modification regulates gene expression and functioning by affecting RNA splicing, stability, translocation, and translation. ObjectiveThis study used comprehensive bioinformatics analysis to elucidate the mechanisms by which m6A regulators function in kidney renal clear cell carcinoma (KIRC) and their interrelationships. MethodsThe KIRC data extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases was used in this study. LASSO Cox regression, protein–protein interaction analysis, univariate Cox regression, and a predictive risk model. Cox regression, Kaplan–Meier analysis, consensus clustering, CIBERSORT, gene set enrichment analysis (GSEA), ESTIMATE, immune cell infiltration, and prognostic factors were investigated in KIRC utilizing computational methods. ResultsThe results of integrated analysis showed that KIRC has unique and complex gene patterns associated with m6A and is linked to different gene patterns. These patterns included an 11.68-fold change in insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3), a 3.35-fold change in ELAV-like RNA binding protein 2 (ELAVL2), a 2.34-fold change in fat mass- and obesity-associated protein (FTO), a 2.52-fold change in ELAVL4, and a 2.15-fold change in ELAVL3. By contrast, there was downregulation of IGF2BP2, zinc finger CCCH-type containing 14 (ZC3H14), and alpha-ketoglutarate-dependent dioxygenase (ALKBH6), with fold changes of 0.44, 0.66, and 0.68, respectively. Minor modifications ranging from 1 to 1.28 were seen in other m6A regulators. ConclusionThis study showed that m6A alteration affects KIRC tumor immunity. Moreover, m6A regulators such as IGF2BP3 have therapeutic and diagnostic potential, which could improve patient outcomes by personalized immunotherapy.

HuR promotes castration-resistant prostate cancer progression by altering ERK5 activation via posttranscriptional regulation of BCAT1

BackgroundCastration-resistant prostate cancer (CRPC) is refractory to hormone treatment, and the underlying mechanism has not been fully elucidated. This study aimed to clarify the role and mechanism of Human antigen R (HuR) as a therapeutic target for CRPC progression.MethodsHuR was knocked out by Cas9 or inhibited by the HuR-specific inhibitor KH-3 in CRPC cell lines and in a mouse xenograft model. The effects of HuR inhibition on tumour cell behaviors and signal transduction were examined by proliferation, transwell, and tumour xenograft assays. Posttranscriptional regulation of BCAT1 by HuR was determined by half-life and RIP assays.ResultsHuR knockout attenuated the proliferation, migration, and invasion of PC3 and DU145 cells in vitro and inhibited tumour progression in vivo. Moreover, BCAT1 was a direct target gene of HuR and mediated the oncogenic effect of HuR on CRPC. Mechanistically, HuR directly interacted with BCAT1 mRNA and upregulated BCAT1 expression by increasing the stability and translation of BCAT1, which activated ERK5 signalling. Additionally, the HuR-specific inhibitor KH-3 attenuated CRPC progression by disrupting the HuR-BCAT1 interaction.ConclusionsWe confirmed that the HuR/BCAT1 axis plays a crucial role in CRPC progression and suggest that inhibiting the HuR/BCAT1 axis is a promising therapeutic approach for suppressing CRPC progression.Graphical

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

FTO ameliorates doxorubicin-induced cardiotoxicity by inhibiting ferroptosis via P53–P21/Nrf2 activation in a HuR-dependent m6A manner

Doxorubicin (DOX)-induced cardiotoxicity seriously limits its clinical applicability, and no therapeutic interventions are available. Ferroptosis, an iron-dependent regulated cell death characterised by lipid peroxidation, plays a pivotal role in DOX-induced cardiotoxicity. N6-methyladenosine (m6A) methylation is the most frequent type of RNA modification and involved in DOX-induced ferroptosis, however, its underlying mechanism remains unclear. P21 was recently found to inhibit ferroptosis by interacting with Nrf2 and is regulated in a P53-dependent or independent manner, such as through m6A modification. In the present study, we investigated the mechanism underlying m6A modification in DOX-induced ferroptosis by focusing on P21. Our results show that fat mass and obesity–associated protein (FTO) down-regulation was associated with DOX-induced cardiotoxicity. FTO over-expression significantly improved cardiac function and cell viability in DOX-treated mouse hearts and H9C2 cells. FTO over-expression significantly inhibited DOX-induced ferroptosis, and the Fer-1 inhibition of ferroptosis significantly reduced DOX-induced cardiotoxicity. P21 was significantly upregulated by FTO and activated Nrf2, playing a crucial role in the anti-ferroptotic effect. FTO upregulated P21/Nrf2 in a P53-dependent manner by mediating the demethylation of P53 or in a P53-independent manner by mediating P21/Nrf2 directly. Human antigen R (HuR) is crucial for FTO-mediated regulation of ferroptosis and P53–P21/Nrf2. Notably, we also found that P21 inhibition in turn inhibited HuR and P53 expression, while HuR inhibition further inhibited FTO expression. RNA immunoprecipitation assay showed that HuR binds to the transcripts of FTO and itself. Collectively, FTO inhibited DOX-induced ferroptosis via P21/Nrf2 activation by mediating the m6A demethylation of P53 or P21/Nrf2 in a HuR-dependent manner and constituted a positive feedback loop with HuR and P53–P21. Our findings provide novel insight into key functional mechanisms associated with DOX-induced cardiotoxicity and elucidate a possible therapeutic approach.

CRISPR/Cas9-Mediated Knockout of the HuR Gene in U251 Cell Inhibits Japanese Encephalitis Virus Replication.

Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus-host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses.

Multifaceted Nature of HuR in Atherosclerosis Development.

HuR (Human antigen R) is an RNA binding protein (RBP) that specifically binds to certain RNA sequences, influencing post-transcriptional regulation. HuR is primarily involved in tumor regulation, as well as cell growth, proliferation, inflammation, and angiogenesis. HuR is implicated in endothelial activation, smooth muscle proliferation, inflammatory response, macrophage apoptosis, lipid regulation, and autophagy, playing a crucial regulatory role in atherosclerosis. Accumulating evidence suggests that HuR has dual roles in AS. On the one hand, HuR expedites the development of AS by facilitating endothelial activation, smooth muscle proliferation, and inflammation. On the contrary, it exerts beneficial effects by reducing macrophage apoptosis, regulating lipid efflux, and increasing autophagy. In this review, we aim to provide a comprehensive summary of the role of HuR in the development of AS by examining its involvement in cellular mechanisms, inflammation, autophagy, and apoptosis. Additionally, we discuss the mechanisms of drugs that target HuR, with the goal of offering new perspectives for the treatment of AS.

UBE4B regulates p27 expression in A549 NSCLC cells through regulating the interaction of HuR and the p27 5’ UTR

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.

A novel NR4A2-HuR axis promotes pancreatic cancer growth and tumorigenesis that is inhibited by NR4A2 antagonists.

Pancreatic ductal adenocarcinoma (PDAC) patients' express higher levels of the orphan Nuclear Receptor 4A2 (NR4A2, NURR1) compared to normal pancreas and NR4A2 is a prognostic factor for patient survival. Knockdown of NR4A2 by RNA interference (RNAi) inhibited cell proliferation, invasion, and migration. RNA sequencing performed in NR4A2(+/+) and NR4A2(-/-) MiaPaCa2 cells demonstrated that NR4A2 played a significant role in cellular metabolism. Human antigen R (HuR) and isocitrate dehydrogenase 1 (IDH1) were identified as NR4A2 target genes. HuR is a pro-oncogenic RNA binding protein and silencing of HuR by RNAi significantly downregulated expression of NR4A2. Expression of HuR and IDH1 were significantly downregulated after treatment with NR4A2 inverse agonist, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane resulting in significant inhibition of tumor growth in an athymic nude mouse xenograft model. This study demonstrates that NR4A2 and HuR regulate genes and signaling pathways that enhance tumorigenesis and targeting NR4A2 and HuR expression with an NR4A2 inverse agonist represents a novel regimen for treating PDAC.