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Xiao Liang,* Yudi Wang,* Hui Shi, Mengmeng Dong, Haobo Han, Quanshun Li Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, People’s Republic of China*These authors contributed equally to this workCorrespondence: Quanshun Li; Haobo Han Tel/ Fax +86-431-85155200Email quanshun@jlu.edu.cn; hanhaobo@jlu.edu.cnBackground: Multidrug resistance (MDR) has emerged to be a major hindrance in cancer therapy, which contributes to the reduced sensitivity of cancer cells toward chemotherapeutic drugs mainly owing to the over-expression of drug efflux transporters. The combination of gene therapy and chemotherapy has been considered as a potential approach to improve the anti-cancer efficacy by reversing the MDR effect.Materials and Methods: The AS1411 aptamer-functionalized micelles were constructed through an emulsion/solvent evaporation strategy for the simultaneous co-delivery of doxorubicin and miR-519c. The therapeutic efficacy and related mechanism of micelles were explored based on the in vitro and in vivo active targeting ability and the suppression of MDR, using hepatocellular carcinoma cell line HepG2 as a model.Results: The micelle was demonstrated to possess favorable cellular uptake and tumor penetration ability by specifically recognizing the nucleolin in an AS1411 aptamer-dependent manner. Further, the intracellular accumulation of doxorubicin was significantly improved due to the suppression of ABCG2-mediated drug efflux by miR-519c, resulting in the efficient inhibition of tumor growth.Conclusion: The micelle-mediated co-delivery of doxorubicin and miR-519c provided a promising strategy to obtain ideal anti-cancer efficacy through the active targeting function and the reversion of MDR.Keywords: micelle, aptamer, nucleolin, multidrug resistance, tumor targeting

Qianhan Lin,1,2,* Xiaoxue Ma,1,2,* Shunxue Hu,3 Rui Li,1,2 Xuan Wei,1,2 Bing Han,1 Yanhui Ma,1 Peishu Liu,1,2,4 Yingxin Pang1,2,4 1Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, People’s Republic of China; 2Key Laboratory of Gynecologic Oncology of Shandong Province, Jinan, 250012, Shandong, People’s Republic of China; 3Department of Pathology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, People’s Republic of China; 4Shandong Engineering Laboratory for Urogynecology, Jinan, 250012, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Peishu LiuDepartment of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, People’s Republic of ChinaTel +8618560081988Email peishuliu@126.comYingxin PangDepartment of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, 250012, Shandong, People’s Republic of ChinaTel +8618560081127Email yingxinpang@126.comPurpose: Nucleolin (NCL) is a multifunctional protein with oncogenic properties. NCL expression levels have been linked to the outcomes of various malignancies, but the clinical value of NCL in patients with endometrial carcinoma (EC) remains unclear. Here, the expression of NCL in EC tissues and its associations with patient outcomes were assessed.Patients and Methods: Data on NCL mRNA expression in EC and adjacent nonneoplastic tissues from The Cancer Genome Atlas (TCGA) were analyzed. In addition, NCL protein expression in 82 endometroid endometrial adenocarcinoma tissues and 15 non-malignant tissues was detected by immunohistochemistry.Results: Elevated NCL expression was markedly correlated with serous endometrial carcinoma (P< 0.001), advanced stage (P=0.029), and grade 3 (P< 0.001). High NCL levels were associated with poorer overall survival (OS) and disease-free survival (DFS) compared with intermediate or low NCL levels (OS: P=0.001, DFS: P=0.006). The multivariate Cox proportional hazards model showed that NCL expression was an independent poor prognostic factor for DFS (HR=1.282, CI=1.027– 1.601, P=0.028). A similar correlation between high expression levels of NCL and unfavorable DFS was found in endometrioid endometrial adenocarcinoma (HR=1.411, CI=1.083– 1.840, P=0.011). Positive extra-nuclear NCL expression (HR=3.377, 95% CI=1.029– 11.186, P=0.046) and low nuclear NCL expression (HR=0.233, 95% CI=0.068– 0.796, P=0.020) were independent prognostic factors for DFS in endometrioid endometrial adenocarcinoma.Conclusion: Heterotopic NCL is a potential prognostic biomarker for EC. Inhibiting the distribution of NCL from the nucleus to the cytoplasm and membrane may be a promising therapeutic strategy to improve outcomes in patients with EC with high NCL expression.Keywords: nucleolin, endometrial carcinoma, prognostic marker, TCGA

The interaction between glioma cells and astrocytes promotes the proliferation of gliomas. Micro-RNAs (miRNAs) carried by astrocyte exosomes (exos) may be involved in this process, but the mechanism remains unclear. The oligonucleotide AS1411, which consists of 26 bases and has a G-quadruplex structure, is an aptamer that targets nucleolin. In this study, we demonstrate exosome-miRNA-27a-mediated cross-activation between astrocytes and glioblastoma and show that AS1411 reduces astrocytes' pro-glioma activity. The enhanced affinity of AS1411 toward nucleolin is attributed to its G-quadruplex structure. After binding to nucleolin, AS1411 inhibits the entry of the NF-κB pathway transcription factor P65 into the nucleus, then downregulates the expression of miRNA-27a in astrocytes surrounding gliomas. Then, AS1411 downregulates astrocyte exosome-miRNA-27a and upregulates the expression of INPP4B , the target gene of miRNA-27a in gliomas, thereby inhibiting the PI3K/AKT pathway and inhibiting glioma proliferation. These results were verified in mouse orthotopic glioma xenografts and human glioma samples. In conclusion, the parallel structure of AS1411 allows it to bind to nucleolin and disrupt the exosome-miRNA-27a-mediated reciprocal activation loop between glioma cells and astrocytes. Our results may help in the development of a novel approach to therapeutic modulation of the glioma microenvironment. [Display omitted] • The exos-miRNA-27a-mediated reciprocal activation loop between glioma and astrocytes. • AS1411 downregulated astrocyte exos-miRNA-27a and upregulated the expression of INPP4B in gliomas. • The enhanced affinity of AS1411 towards nucleolins attributed to its G-quadruplex parallel structure characteristic. [ABSTRACT FROM AUTHOR]

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell. [Display omitted] [ABSTRACT FROM AUTHOR]

A burgeoning topic in the early diagnosis and treatment of cancer is the sensitive and precise quantification of circulating tumor cells (CTCs). However, due to the extremely low CTC concentrations in the blood, traditional enrichment and identification methods for CTCs are prone to false negative results. Here, to specifically identify and improve the sensitivity of CTC detection in the blood, we designed a novel fluorometric turn-on aptasensor platform based on a three-dimensional network nanovehicle (TDNN) modified with a PEG derivative and AS1411 aptamer specifically targeting the nucleolin protein. The limit of detection (LOD) of the aptasensor platform was as low as 2 cells per mL, which was remarkably better than that of the single nanoparticle ASS1. Notably, the novel aptasensor platform showed excellent specificity and sensitivity as well as negligible fluorescent background for quantitative and visualization detection of CTCs in peripheral blood samples without enrichment, centrifugation and washing steps. More importantly, the number of CTCs identified by TDNN was significantly higher in metastatic patients than in nonmetastatic patients, while CTCs were not detected in the blood samples of healthy donors. In conclusion, the aptasensor platform provides an available and promising tool for CTC identification and cancer metastasis prediction in clinical blood samples. • We develop a novel aptasensor platform for ultrasensitive detection of cancer cells. • The detection limit was as low as 2 cell/mL. • The aptasensor platform has high sensitivity, high selectivity, excellent stability, and anti-interference ability. • The aptasensor platform were successfully used for CTC detection in clinical trials. • This detection method does not involve any enrichment or extraction of nucleic acids and proteins. [ABSTRACT FROM AUTHOR]

Hypoxia induces the abnormal proliferation of vascular smooth muscle cells (VSMCs), resulting in the pathogenesis of various vascular diseases. RNA-binding proteins (RBPs) are involved in a wide range of biological processes, including cell proliferation and responses to hypoxia. In this study, we observed that the RBP nucleolin (NCL) was downregulated by histone deacetylation in response to hypoxia. We evaluated its regulatory effects on miRNA expression under hypoxic conditions in pulmonary artery smooth muscle cells (PASMCs). miRNAs associated with NCL were assessed using RNA immunoprecipitation in PASMCs and small RNA sequencing. The expression of a set of miRNAs was increased by NCL but reduced by hypoxia-induced downregulation of NCL. The downregulation of miR-24-3p and miR-409-3p promoted PASMC proliferation under hypoxic conditions. These results clearly demonstrate the significance of NCL–miRNA interactions in the regulation of hypoxia-induced PASMC proliferation and provide insight into the therapeutic value of RBPs for vascular diseases.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocytophilum protein) and found that it dynamically changed its pattern and subcellular location in cells and enhanced cell adhesion. Tandem affinity purification combined with mass spectrometry identified host nucleolin as an AFAP-interacting protein. Further study showed the disruption of nucleolin by RNA interference, and the treatment of a nucleolin-binding DNA aptamer AS1411 attenuated AFAP-mediated cell adhesion, indicating that AFAP enhanced cell adhesion in a nucleolin-dependent manner. The characterization of cell adhesion-enhancing AFAP and the identification of host nucleolin as its interaction partner may help understand the mechanism underlying A. phagocytophilum-promoting cell adhesion, facilitating the elucidation of HGA pathogenesis.

Objective: Ovarian high-grade serous carcinoma (HGSC) is a fatal malignancy of women. Alterations in the expression of nuclear proteins are early steps in malignant transformation; nucleolin is one such protein. Changes in nucleolin expression and circulatory levels during ovarian HGSC development are unknown. The study goal was to determine if tissue and circulatory levels of nucleolin change in response to malignant transformation leading to ovarian HGSC. Methods: Sera, ovaries, and BRCA+ fimbria from healthy subjects, and sera and tumor tissues from patients (n = 10 each), and healthy hens and hens with HGSC were examined in exploratory and prospective studies for nucleolin expression by immunohistochemistry, immunoblotting, gene expression, and immunoassay, and analyzed by analysis of variance (ANOVA). Results: Compared with normal, nucleolin expression was higher in patients and hens with ovarian HGSC and in women with a risk of HGSC (P < 0.05). Compared with normal (1400 + 105 pg/mL, n = 8), serum nucleolin levels were 1.5 and 1.7-fold higher in patients with early- (n = 5) and late-stage (n = 5) HGSC, respectively. Additionally, serum nucleolin levels increased significantly (P < 0.05) prior to the formation of detectable masses. Conclusion: This pilot study concluded that tissue and serum levels of nucleolin increase in association with malignant changes in ovaries and fimbriae leading to ovarian HGSC.

α-Synuclein (αSyn) is an important player in Parkinson’s disease (PD) pathogenesis. The aggregation of αSyn is mainly formed in the cytoplasm, whereas some αSyn accumulation has also been found in the nuclei of neurons. To assess the effect of nuclear αSyn, we generated αSyn conjugated with a nuclear export signal (NES) or a nuclear localization signal (NLS), and compared them with wild-type αSyn in primary mouse embryonic fibroblasts (MEF) using DNA transfection. Overexpression of NLS-αSyn increased cytotoxicity. The levels of apoptotic markers were increased by NLS-αSyn in MEF. Interestingly, an increase in the levels of 40S ribosomal protein 15 was observed in MEF expressing NLS-αSyn. These MEF also showed a higher 28S/18S rRNA ratio. Intriguingly, the expression of NLS-αSyn in MEF enhanced segmentation of nucleolin (NCL)-positive nucleolar structures. We also observed that the downregulation of NCL, using shRNA, promoted a relatively higher 28S/18S rRNA ratio. The reduction in NCL expression accelerated the accumulation of αSyn, and NCL transfection enhanced the degradation of αSyn. These results suggest that nuclear αSyn contributes to the alteration in ribosomal RNA processing via NCL malfunction-mediated nucleolar segmentation, and that NCL is a key factor for the degradation of αSyn.

Background: The nucleolus is a subnuclear, non-membrane bound domain that is the hub of ribosome biogenesis and a critical regulator of cell homeostasis. Rapid growth and division of cells in tumors are correlated with intensive nucleolar metabolism as a response to oncogenic factors overexpression. Several members of the Epidermal Growth Factor Receptor (EGFR) family, have been identified in the nucleus and nucleolus of many cancer cells, but their function in these compartments remains unexplored. Results: We focused our research on the nucleolar function that a specific member of EGFR family, the ErbB3 receptor, plays in glioblastoma, a tumor without effective therapies. Here, Neuregulin 1 mediated proliferative stimuli, promotes ErbB3 relocalization from the nucleolus to the cytoplasm and increases pre-rRNA synthesis. Instead ErbB3 silencing or nucleolar stress reduce cell proliferation and affect cell cycle progression. Conclusions: These data point to the existence of an ErbB3-mediated non canonical pathway that glioblastoma cells use to control ribosomes synthesis and cell proliferation. These results highlight the potential role for the nucleolar ErbB3 receptor, as a new target in glioblastoma. [ABSTRACT FROM AUTHOR]

Monitoring the cell surface-expressed nucleolin facilitates early cancer diagnosis. Herein, we developed a multivalent aptamer displacement strand duplex strategy on cell membranes using a multi-receptor co-recognition design for improving the sensitivity and specificity of cancer cell recognition with an ultra-low background. The AS1411 aptamer labeled with the FAM fluorophore can be quenched using a partial complementary sequence modified with a BHQ1 tag which is partially hybridized with the AS1411 aptamer to create a receptor-activating aptamer. The multi-AS1411 activable probe based on the strand displacement strategy was constructed using multiple copies of the structure-switching AS1411 aptamer (bearing a short poly-A tail) linked together using the poly-T long chain (as a scaffold) which was synthesized by Terminal Deoxynucleotidyl Transferase (TDT)-mediated extension. We demonstrated the promising efficacy and sensitivity of our method in recognizing tumor cells in both cell mixtures and clinical cytology specimens. Due to its simple and fast operation with excellent cell recognition sensitivity and accuracy, it is expected to achieve the detection of low abundance target cells. Our approach will have broad application in clinical rapid detection and personalized medicine. [ABSTRACT FROM AUTHOR]

Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of initiator-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs. [ABSTRACT FROM AUTHOR]

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)‐mediated antiviral responses, including through inhibition of DNA‐binding by IFN‐activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non‐pathogenic Ni‐derived strain, Ni‐CE, differs significantly, with nuclear accumulation defective for Ni‐CE‐P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine‐bovine (B‐B), ovine‐ovine (O‐O) SCNT, and ovine‐bovine (O‐B) iSCNT embryos and found that a developmental block existed in the 8‐cell stage in O‐B iSCNT embryos. RNA sequencing and q‐PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O‐B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O‐B iSCNT embryos. In addition, the nucleolin exhibited a clear circular‐ring structure in B‐B 8‐cell stage embryos, whereas this was shell‐like or dot‐like in the O‐B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring‐like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell‐like nucleolin‐structure in B‐B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos. [ABSTRACT FROM AUTHOR]

We aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation assays. Western-blot and q-PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP-deficient plasmid of ARHGAP30R55A. Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL. [ABSTRACT FROM AUTHOR]

In all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2ʹ-O-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB). Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2ʹ-O-methylation sites in A. thaliana Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2ʹ-O-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2ʹ-O-methylation at specific rRNA sites suggesting functional/structural interconnections of 2ʹ-O-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plants. [ABSTRACT FROM AUTHOR]

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein‐protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid‐liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA‐RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease. SYNOPSIS: Nucleolin is a multifunctional RNA Binding Protein with diverse subcellular localizations. Here we show that the glycine arginine rich (GAR) domain drives localization of nucleolin via direct interaction with a kinesin light chain and plasma membrane association of arginine rich sequences. Nucleolin GAR domain binds a kinesin light chain, directly linking nucleolin‐mRNA complexes to kinesin motors for axonal transport.Heterozygous nucleolin GAR deletion neurons reveal deficits in axonal localization of nucleolin cargo mRNAs.Heterozygous nucleolin GAR deletion neurons reveal increased axonal outgrowth.The GAR domain is also required for membrane targeting of nucleolin. [ABSTRACT FROM AUTHOR]

Infection with Helicobacter pylori (H. pylori) leads to a fork in the road situation where it is critical and complex to judge the fate of the cell. We propose for the first time an in silico representation of a protein level network model that can unfold the mystery behind the cell fate decision between inflammation or cell proliferation or cell death. Upon infection TNF inducible protein α (Tip α) is internalised after binding with the cell surface receptor Nucleolin which is overexpressed on the cell surface thereby activating the Ras pathway. Tip α, Nucleolin and Ras decides the cell fate for apoptosis or abnormal cell proliferation along with ulcers in the gastric tract, hence we term it as the "death triad", which otherwise triggers the inflammatory pathway through downstream signalling of NF-κβ. A series of proteins involved in the signalling cascade are portrayed through compartmentalization of the bacteria and the gut wall. The depicted network works synchronously toward an overarching goal of deciding between apoptosis or inflammation or proliferation. The model has been validated by simulating it with existing transcriptomic data along with clinical findings from patients infected with H. pylori across different regions in India. The results clearly indicate that for a short period of time there is increased binding of Tip α to Nucleolin and the receptor starts to saturate. This increases the tenacity of binding and the cell triggers an inflammatory cascade reaction which involves proinflammatory cytokines such as TNF α thereby progressing to inflammation by activating NF-κβ downstream. On the other hand, Ras involved in interaction with nucleolin can be present both in its activated or inactivated state. Binding of Tip α as a monomer leads to desensitization of Nucleolin leading to cell survival and proliferation. [ABSTRACT FROM AUTHOR]

This work was aimed at studying the structural organization of nucleolus and constitutive heterochromatin in different types of tanycytes during postnatal development and aging of rats. The distribution of nucleolus argentophilic proteins (nucleolin and nucleophosmin) and heterochromatin aggregates in tanycytes at various stages of postnatal development have been described for the first time using immunohistochemical methods and confocal laser microscopy. The heterogeneity of the size and number of nucleoli was demonstrated both in different tanycytes subpopulations and at different ages of an animal. This may indicate different levels of the tanycyte synthetic activity and the ability to proliferate during early postnatal development and aging. During aging, the distribution of heterochromatin aggregates varies among tanycyte subpopulations: α-tanycytes undergo intense heterochromatization, while β-tanycytes are characterized by a stable organization of the studied compartments of the cell nucleus. The data obtained significantly supplement the modern understanding of organization of the structure of the cell nucleus of tanycytes during normal development and aging. This can subsequently serve as a basis for establishing the role of these subnuclear structures in pathological processes. [ABSTRACT FROM AUTHOR]

Background Visceral pleural invasion (VPI) in adenocarcinoma of the lung is considered a poor prognostic factor. The purpose of this study was to analyze nucleolin and nucleophosmin expression in pulmonary adenocarcinoma (PA) with VPI and in pleural malignant mesothelioma. Methods The study was conducted on the basis of 19 pathologically‐confirmed cases of adenocarcinoma of the lung and 29 cases of epithelioid malignant mesothelioma. The nucleolin and nucleophosmin expression was assessed immunohistochemically and analyzed with image analysis software. Results Nucleolin expression was lower while nucleophosmin was higher in pleural invasion of pulmonary adenocarcinoma than in the central part of the tumor. Differences in subpopulations of cells with different expression of proteins studied were also found. Malignant mesothelioma showed lower nucleolin expression than adenocarcinoma of the lung but no differences in nucleophosmin expression were found. Conclusions The results of our study suggested that lower nucleolin and higher nucleophosmin expression may be related to higher invasiveness of adenocarcinoma of the lung. Differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma indicate another aspect of biology of these pleura‐invading cancers that requires further study. Key points Significant findings of the study Differences in nucleolin and nucleophosmin expression in pleura invading pulmonary adenocarcinoma indicate the involvement of these proteins in its locoregional spread while differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma suggest another aspect of biology of these cancers. What this study adds This is the first study on nucleolin and nucleophosmin expression in pleural malignant mesothelioma and pleura‐invading pulmonary adenocarcinoma. Our findings may assist in understanding the mechanisms of locoregional spread of adenocarcinoma and differences between these two pleura‐invading cancers.

Abstract Background Hepatoma-derived growth factor (HDGF) participates in angiogenesis and represents a negative prognostic factor in oral cancer. The current study was designed to elucidate the regulatory mechanism between HDGF and vascular endothelial growth factor (VEGF) and the clinical impact of oral cancer. Methods TCGA data and surgical samples from oral cancer patients were used for the clinicopathological parameter and survival analysis. Human oral cancer SCC4 and SAS cells were treated with recombinant HDGF protein. VEGF gene expression and protein level were analyzed by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. The signaling pathways for regulating VEGF expression were investigated. The nucleolin neutralizing antibody and HIF-1α inhibitor were applied to SCC4 cells to investigate their effects on the HDGF-stimulated VEGF pathways. Results TCGA and immunohistochemical analysis revealed a positive correlation between HDGF and VEGF expression in oral cancer tissues. Recombinant HDGF significantly increased VEGF gene and protein expression in oral cancer SCC4 cells in a dose-dependent manner. HDGF enhanced the phosphorylation levels of AKT and IkB and the protein level of HIF-1α and NF-κB. The nucleolin-neutralizing antibody abolished HDGF-stimulated HIF-1α, NF-κB and VEGF protein expression in SCC4 cells. The HIF-1α inhibitor antagonized the HDGF-induced VEGF gene expression. High VEGF expression was strongly correlated with HDGF expression, advanced disease, and poor survival. Conclusion This study postulated a new pathway in which HDGF activated HIF-1α and then induced VEGF expression through binding to membrane nucleolin under normoxic conditions, leading to poor disease control. The HDGF/HIF-1α/VEGF axis is important for developing future therapeutic strategies.

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl–NCL–VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO 2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn 2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn 2+ -mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Rationale: Myocardial infarction (MI) is a severe global clinical condition with widespread prevalence. The adult mammalian heart's limited capacity to generate new cardiomyocytes (CMs) in response to injury remains a primary obstacle in developing effective therapies. Current approaches focus on inducing the proliferation of existing CMs through cell-cycle reentry. However, this method primarily elevates cyclin dependent kinase 6 (CDK6) and DNA content, lacking proper cytokinesis and resulting in the formation of dysfunctional binucleated CMs. Cytokinesis is dependent on ribosome biogenesis (Ribo-bio), a crucial process modulated by nucleolin (Ncl). Our objective was to identify a novel approach that promotes both DNA synthesis and cytokinesis. Methods: Various techniques, including RNA/protein-sequencing analysis, Ribo-Halo, Ribo-disome, flow cytometry, and cardiac-specific tumor-suppressor retinoblastoma-1 (Rb1) knockout mice, were employed to assess the series signaling of proliferation/cell-cycle reentry and Ribo-bio/cytokinesis. Echocardiography, confocal imaging, and histology were utilized to evaluate cardiac function. Results: Analysis revealed significantly elevated levels of Rb1, bur decreased levels of circASXL1 in the hearts of MI mice compared to control mice. Deletion of Rb1 induces solely cell-cycle reentry, while augmenting the Ribo-bio modulator Ncl leads to cytokinesis. Mechanically, bioinformatics and the loss/gain studies uncovered that circASXL1/CDK6/Rb1 regulates cell-cycle reentry. Moreover, Ribo-Halo, Ribo-disome and circRNA pull-down assays demonstrated that circASXL1 promotes cytokinesis through Ncl/Ribo-bio. Importantly, exosomes derived from umbilical cord mesenchymal stem cells (UMSC-Exo) had the ability to enhance cardiac function by facilitating the coordinated signaling of cell-cycle reentry and Ribo-bio/cytokinesis. These effects were attenuated by silencing circASXL1 in UMSC-Exo. Conclusion: The series signaling of circASXL1/CDK6/Rb1/cell-cycle reentry and circASXL1/Ncl/Ribo-bio/cytokinesis plays a crucial role in cardiac repair. UMSC-Exo effectively repairs infarcted myocardium by stimulating CM cell-cycle reentry and cytokinesis in a circASXL1-dependent manner. This study provides innovative therapeutic strategies targeting the circASXL1 signaling network for MI and offering potential avenues for enhanced cardiac repair., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors., Competing Interests: Conflict of Interest Statement DP and CMC are inventors of the patent application WO2017011411A1 (methods and compositions relating to anti-NCL recombinant immunoagents). DP and TJM are consultants and own equity Koru Biopharma, which is developing anti-NCL agents.

Despite many advances in the use of DNA nanodevices as assembly or disassembly modules to build various complex structures, the simultaneous assembly and disassembly of DNA structures in living cells remains a challenge. In this study, we present a modular engineering approach for assembling and disassembling DNA nanodevices in response to endogenous biomarkers. As a result of pairwise prehybridization of original DNA strands, the DNA nanodevice is initially inert. In an effort to bind one of the paired strands and release its complement, nucleolin competes. Assembly of the DNA nanodevice is initiated when the released complement binds to it, and disassembly is initiated when APE1 shears the assembled binding site of the DNA nanodevice. Spatial-temporal logic control is achieved through our approach during the assembly and disassembly of DNA nanodevices. Furthermore, by means of this assembly and disassembly procedure, the sequential detection and imaging of two tumor markers can be achieved, thereby effectively reducing false-positive signal results and accelerating the detection time. This study emphasizes the simultaneous assembly and disassembly of DNA nanodevices controlled by biomarkers in a simple and versatile manner; it has the potential to expand the application scope of DNA nanotechnology and offers an idea for the implementation of precision medicine testing.

Targeted nanodelivery systems offer a promising approach to cancer treatment, including the most common cancer in women, breast cancer. In this study, a targeted, pH-responsive, and biocompatible nanodelivery system based on nucleolin aptamer-functionalized biogenic titanium dioxide nanoparticles (TNP) was developed for targeted co-delivery of FOXM1 aptamer and doxorubicin (DOX) to improve breast cancer therapy. The developed targeted nanodelivery system exhibited almost spherical morphology with 124.89 ± 12.97 nm in diameter and zeta potential value of - 23.78 ± 3.66 mV. FOXM1 aptamer and DOX were loaded into the nanodelivery system with an efficiency of 100% and 97%, respectively. Moreover, the targeted nanodelivery system demonstrated excellent stability in serum and a pH-responsive sustained drug release profile over a period of 240 h following Higuchi kinetic and Fickian diffusion mechanism. The in vitro cytotoxicity experiments demonstrated that the targeted nanodelivery system provided selective internalization and strong growth inhibition effects of about 45 and 51% against nucleolin-positive 4T1 and MCF-7 breast cancer cell lines. It is noteworthy that these phenomena were not observed in nucleolin-negative cells (CHO). The preclinical studies revealed that a single-dose intravenous injection of the targeted nanodelivery system into 4T1-bearing mice inhibited tumor growth by 1.7- and 1.4-fold more efficiently than the free drug and the non-targeted nanodelivery system, respectively. Our results suggested that the developed innovative targeted pH-responsive biocompatible nanodelivery system could serve as a prospectively potential platform to improve breast cancer treatment., (© 2023. Controlled Release Society.)

DNA is an excellent programmable polymer for the generation of self-assembled multivalent nanostructures useful for biomedical applications. Herein, we developed (i) folate-functionalized nanocages (Fol-NC), very efficiently internalized by tumor cells overexpressing the α isoform of the folate receptor; (ii) AS1411-linked nanocages (Apt-NC), internalized through nucleolin, a protein overexpressed in the cell surface of many types of cancers; and (iii) nanostructures that harbor both folate and AS1411 aptamer functionalization (Fol-Apt-NC). We analyzed the specific miRNA silencing activity of all types of nanostructures harboring miRNA sequestering sequences complementary to miR-21 and the cytotoxic effect when loaded with doxorubicin in a drug-resistant triple-negative breast cancer cell line. We demonstrate that the presence of folate as a targeting ligand increases the efficiency in miR-21 silencing compared to nanocages functionalized with AS1411. Double-functionalized nanocages (Fol-Apt-NC), loaded with doxorubicin, resulted in an increase of over 51% of the cytotoxic effect on MDA-MB-231 cells compared to free doxorubicin, demonstrating, besides selectivity, the ability of nanocages to overcome Dox chemoresistance. The higher efficiency of the folate-functionalized nanocages is due to the way of entrance, which induces more than four times higher intracellular stability and indicates that the folate-mediated route of cell entry is more efficient than the nucleolin-mediated one when both folate and AS1411 modifications are present.

In this paper, we study the biological properties of two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, respectively, and two further derivatives in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics. The therapeutical development of the TBA was hindered by its low thermodynamic and nuclease stability, while its potential as an anticancer/antiproliferative molecule is also affected by the anticoagulant activity, being a side effect in this case. In order to obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of the above described TBA derivatives have been tested. Interestingly, none of the TBA analogs exhibits an anticoagulant activity, while all of them show antiproliferative properties to the same extent. Furthermore, TBAG4 displays extraordinary nuclease stability and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer and antiviral drugs.

Background: Sepsis-induced myocardial injury, as one of the important complications of sepsis, can significantly increase the mortality of septic patients. Our previous study found that nucleolin affected mitochondrial function in energy synthesis and had a protective effect on septic cardiomyopathy in mice. During sepsis, glucose metabolism disorders aggravated myocardial injury and had a negative effect on septic patients., Objectives: We investigated whether nucleolin could regulate glucose metabolism during endotoxemia-induced myocardial injury., Methods: The study tested whether the nucleolin cardiac-specific knockout in the mice could affect glucose metabolism through untargeted metabolomics, and the results of metabolomics were verified experimentally in H9C2 cells. The ATP content, lactate production, and oxygen consumption rate (OCR) were evaluated., Results: The metabolomics results suggested that glycolytic products were increased in endotoxemia-induced myocardial injury, and that nucleolin myocardial-specific knockout altered oxidative phosphorylation-related pathways. The experiment data showed that TNF-α combined with LPS stimulation could increase the lactate content and the OCR values by about 25%, and decrease the ATP content by about 25%. However, interference with nucleolin expression could further decrease ATP content and OCR values by about 10-20% and partially increase the lactate level in the presence of TNF-α and LPS. However, nucleolin overexpression had the opposite protective effect, which partially reversed the decrease in ATP content and the increase in lactate level., Conclusion: Down-regulation of nucleolin can exacerbate glucose metabolism disorders in endotoxemia-induced myocardial injury. Improving glucose metabolism by regulating nucleolin was expected to provide new therapeutic ideas for patients with septic cardiomyopathy., Competing Interests: The authors declare that they have no competing interests., (© 2024 Tang et al.)

We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Nowak, Lenartowski, Kalita, Lehka, Karatsai, Lenartowska and Rędowicz.)

We fabricated hybrid nanoparticles consisting of organic semiconducting material with peptide sequence to reflect the target protein interaction. A phosphorescent OLED material, platinum octaethylporphyrin (PtOEP) was self-assembled by reprecipitation with the A17 peptide (YCAYYSPRHKTTF) selected as a probe ligand in order to recognize heat shock protein 70 (HSP70). The phosphorescence intensity of the PtOEP-A17 assembly was enhanced by 125 % after treatment with HSP70. The specificity of the protein interaction was confirmed in both solution and solid states of the PtOEP-A17 assembly against to BSA and nucleolin. We figured out that the phosphorescence lifetime of PtOEP-A17 assembly after exposed to HSP70 increased significantly to 153 ns from initial 115 ns. These simultaneous enhancements in phosphorescence and lifetime triggered by the specific protein interaction would open new applications of PtOEP, a representative material of light-emitting device fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5'-TTG GGT GGG GAA-3'), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and a computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs.

Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10–30 μM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 μM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10–30 μM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.

SOX2, a member of the SRY-related HMG-box (SOX) family, is abnormally expressed in many tumors and associated with cancer stem cell-like properties. Previous reports have shown that SOX2 is a biomarker for cancer stem cells in human bladder cancer (BC), and our most recent study has indicated that the inhibition of SOX2 by anticancer compound ChlA-F attenuates human BC cell invasion. We now investigated the mechanisms through which SOX2 promotes the invasive ability of BC cells. Our studies revealed that SOX2 promoted SKP2 transcription and increased SKP2-accelerated Sp1 protein degradation. As Sp1 is a transcriptionally regulated gene, HUR transcription was thereby attenuated, and, in the absence of HUR, FOXO1 mRNA was degraded fast, which promoted BC cell invasion. In addition, SOX2 promoted BC invasion through the upregulation of nucleolin transcription, which resulted in increased MMP2 mRNA stability and expression. Collectively, our findings show that SOX2 promotes BC invasion through both SKP2-Sp1-HUR-FOXO1 and nucleolin-MMP2 dual axes.

Gene therapy is a promising approach for treating tumors. Conventional approaches of DNA delivery depending on non-viral or viral vectors are unsatisfactory due to the concerns of biosafety and cell-targeting efficiency. The question how to deliver DNA into tumor cells efficiently and selectively is a major technological problem in tumor gene therapy. Here, we develop a vector-free gene transfer strategy to deliver genes effectively and selectively by taking advantage of targeting nucleolin. Nucleolin, a shuttle protein moving between cell membrane, cytoplasm and nuclei, is overexpressed in tumor cells. It has a natural ligand G-quadruplex (Gq). Gq-linked DNA (Gq-DNA) is likely to be internalized by ligand dependent uptake mechanisms independently of vectors after neutralizing negative charges of cell membrane by targeting nucleolin. This strategy is referred to as Gq-DNA transfection. Benefiting from its high affinity to nucleolin, Gq-DNA can be effectively delivered into nucleolin-positive tumor cells even nuclei. Gq-DNA transfection is characterized by low cytotoxicity, high efficiency, ease of synthesis, high stability in serum, direct access into nuclei, and specific nucleolin-positive tumor cell targeting.

Post-operative chemotherapy is still required for the treatment of glioblastoma (GBM), for which nanocarrier-based drug delivery has been identified as one of the most effective methods. However, the blood-brain barrier (BBB) and non-specific delivery to non-tumor tissues can significantly limit drug accumulation in tumor tissues and cause damage to nearby normal tissues. This study describes a targeted cancer therapy approach that uses AS1411 aptamer-conjugated nanospheres (100-300 nm in size) loaded with doxorubicin (Dox) to selectively identify tumor cells overexpressing nucleolin (NCL) proteins. The study demonstrates that the active target model, which employs aptamer-mediated drug delivery, is more effective than non-specific enhanced permeability and maintenance (EPR)-mediated delivery and passive drug delivery in improving drug penetration and maintenance in tumor cells. Additionally, the study reveals the potential for anti-cancer effects through 3D spheroidal and in vivo GBM xenograft models. The DNA-protein hybrid nanospheres utilized in this study offer numerous benefits, such as efficient synthesis, structural stability, high drug loading, dye labeling, biocompatibility, and biodegradability. When combined with nanospheres, the 1411 aptamer has been shown to be an effective drug delivery carrier allowing for the precise targeting of tumors. This combination has the potential to produce anti-tumor effects in the active targeted therapy of GBM.

Previous research results of our group showed that Toll-like receptor 4 (TLR4) and nucleolin synergistically mediate respiratory syncytial virus (RSV) infection in human central neuron cells, but the specific mechanism remains unclear. Here we designed and synthesized lentiviruses with TIR (674-815 aa), TLR4 (del 674-815 aa), GAR (645-707 aa), and NCL (del 645-707 aa) domains, and obtained stable overexpression cell lines by drug screening, and subsequently infected RSV at different time points. Laser confocal microscopy and coimmunoprecipitation were used for the observation of co-localization and interaction of TIR/GAR domains. Western blot analysis was used for the detection of p-NF-κB and LC3 protein expression. Real-time PCR was used for the detection of TLR4/NCL mRNA expression. ELISA assay was used to measure IL-6, IL-1β, and TNF-α concentrations and flow cytometric analysis was used for the study of apoptosis. Our results suggest that overexpression of TIR and GAR domains can exacerbate apoptosis and autophagy, and that TIR and GAR domains can synergistically mediate RSV infection and activate the NF-κB signaling pathway, which regulates the secretion of downstream inflammatory factors, such as IL-6, IL-1β, and TNF-α, and ultimately leads to neuronal inflammatory injury., (© 2024 Wiley Periodicals LLC.)

Recent studies suggest that circular RNA (circRNA)-mediated post-translational modification of RNA-binding proteins (RBP) plays a pivotal role in metastasis of hepatocellular carcinoma (HCC). However, the specific mechanism and potential clinical therapeutic significance remain vague. This study attempts to profile the regulatory networks of circRNA and RBP using a multi-omics approach. Has&#95;circ&#95;0006646 (circ0006646) is an unreported circRNA in HCC and is associated with a poor prognosis. Silencing of circ0006646 significantly hinders metastasis in vivo. Mechanistically, circ0006646 prevents the interaction between nucleolin (NCL) and the E3 ligase tripartite motif-containing 21 to reduce the proteasome-mediated degradation of NCL via K48-linked polyubiquitylation. Furthermore, the change of NCL expression is proven to affect the phosphorylation levels of multiple proteins and inhibit p53 translation. Moreover, patient-derived tumor xenograft and lentivirus injection, which is conducted to simulate clinical treatment confirmed the potential therapeutic value. Overall, this study describes the integrated multi-omics landscape of circRNA-mediated NCL ubiquitination degradation in HCC metastasis and provides a novel therapeutic target., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

In this issue of Molecular Cell , Liu et al. (2022) report that 5′-tRFCys, a stress-induced transfer RNA-derived RNA fragment (tRF) derived from the 5′ halves of cysteine tRNAs, regulates post-transcriptional gene expression, enabling the survival and lung metastasis formation of breast cancers. [ABSTRACT FROM AUTHOR]

Background: Polysialic acid (polySia) modifies six cell surface proteins in humans mainly during fetal development and some blood cells in adults. Two genes in humans, ST8SIA2 and ST8SIA4, code for polysialyltransferases that synthesize polySia. ST8SIA2 is highly expressed during fetal development and in cancer but not in adult normal human cells. ST8SIA4 is expressed in fetal and adult brain, spleen, thymus, and peripheral blood leukocytes and in cancer. We identified a derivative of polySia containing de-N-acetyl neuraminic acid residues (dPSA), which is expressed on the cell surface of human cancer cell lines and tumors but not normal cells. Methods: dPSA-modified proteins in several human cancer cell lines and normal blood cells were identified using co-immunoprecipitation with anti-dPSA antibodies, mass spectroscopy and Western blot. RNAi and CRISPR were used to knockdown and knockout, respectively, the polysialyltransferase genes in human melanoma SK-MEL-28 and neuroblastoma CHP-134 cell lines, respectively, to determine the effect on production of cell surface dPSA measured by flow cytometry and fluorescence microscopy. Results: We found that dPSA is linked to or associated with nucleolin, a nuclear protein reported to be on the cell surface of cancer but not normal cells. Knocking down expression of ST8SIA2 with RNAi or knocking out each gene individually and in combination using CRISPR showed that cell surface dPSA depended on expression of ST8SIA2. Conclusions: The presence of dPSA specifically in a broad range of human cancers but not human adult normal cells offers novel possibilities for diagnosis, prevention and treatment targeting the dPSA antigen that appears to be cancer-specific, consistent across not only human cancers but also species, and may be an unrecognized mechanism of immune shielding. [ABSTRACT FROM AUTHOR]

Cancer is the second leading cause of death worldwide after cardiovascular disease. Depending on the type and the location of the tumor, several cancer treatments are implemented. Among these, the three most conventional therapies are surgery, radiotherapy and chemotherapy. However, there are other therapeutic approaches such as photodynamic therapy (PDT). PDT relies on the combined action of light, a photoactivable molecule called photosensitizer (PS) and molecular oxygen. Most of the PSs used for clinical applications are not cancer-cell specific. One of the solutions to overcome this problem is the use of nanoparticles (NPs) to induce a passive targeting. It is also possible to graft a vector onto the NPs to specifically target membrane receptors overexpressed in the tumor cells or neovessels surrounding the tumor. In this review, we focus on the NPs loaded with PSs and coupled to peptides for targeted PDT. We described nanosystems that targeted Neuropilin-1 (NRP-1), αvβ3 integrins, nucleolin membrane receptor, epidermal growth factor (EGF) receptor, protein-glutamine-gamma-glutamyltransferase (TGM2), p32, transferrin, PD-1, and mitochondrial membrane. The use of a cell absorbing-peptide is also described. [ABSTRACT FROM AUTHOR]

Premise: Light and gravity are fundamental cues for plant development. Our understanding of the effects of light stimuli on plants in space, without gravity, is key to providing conditions for plants to acclimate to the environment. Here we tested the hypothesis that the alterations caused by the absence of gravity in root meristematic cells can be counteracted by light. Methods: Seedlings of wild‐type Arabidopsis thaliana and two mutants of the essential nucleolar protein nucleolin (nuc1, nuc2) were grown in simulated microgravity, either under a white light photoperiod or under continuous darkness. Key variables of cell proliferation (cell cycle regulation), cell growth (ribosome biogenesis), and auxin transport were measured in the root meristem using in situ cellular markers and transcriptomic methods and compared with those of a 1 g control. Results: The incorporation of a photoperiod regime was sufficient to attenuate or suppress the effects caused by gravitational stress at the cellular level in the root meristem. In all cases, values for variables recorded from samples receiving light stimuli in simulated microgravity were closer to values from the controls than values from samples grown in darkness. Differential sensitivities were obtained for the two nucleolin mutants. Conclusions: Light signals may totally or partially replace gravity signals, significantly improving plant growth and development in microgravity. Despite that, molecular alterations are still compatible with the expected acclimation mechanisms, which need to be better understood. The differential sensitivity of nuc1 and nuc2 mutants to gravitational stress points to new strategies to produce more resilient plants to travel with humans in new extraterrestrial endeavors. [ABSTRACT FROM AUTHOR]

Conventional chemotherapy used against cancer is mostly limited due to their non-targeted nature, affecting normal tissue and causing undesirable toxic effects to the affected tissue. With the aim of improving these treatments both therapeutically and in terms of their safety, numerous studies are currently being carried out using nanoparticles (NPs) as a vector combining tumor targeting and carrying therapeutic tools. In this context, it appears that nucleolin, a molecule over-expressed on the surface of tumor cells, is an interesting therapeutic target. Several ligands, antagonists of nucleolin of various origins, such as AS1411, the F3 peptide and the multivalent pseudopeptide N6L have been developed and studied as therapeutic tools against cancer. Over the last ten years or so, numerous studies have been published demonstrating that these antagonists can be used as tumor targeting agents with NPs from various origins. Focusing on nucleolin ligands, the aim of this article is to review the literature recently published or under experimentation in our research team to evaluate the efficacy and future development of these tools as anti-tumor agents. [ABSTRACT FROM AUTHOR]

Background Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. Results We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) ≤ 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing ≤ 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p < 0.01). Compared with normal B-cells, CLL cells over-expressed thyroid hormone receptor-associated protein 3 (THRAP3; n = 9; p = 0.00007), which is implicated in cell proliferation; and heterochromatin protein 1-binding protein 3 (HP1BP3; n = 10; p = 0.0002), which promotes cell survival and tumourogenesis. A smaller form of HP1BP3, which may correspond to HP1BP3 isoform-2, was specifically identified in normal B-cells (n = 10; p = 0.0001). HP1BP3 and THRAP3 predicted poor prognosis of CLL (p ≤ 0.05). Consistently, THRAP3 and HP1BP3 were found to be associated with cancer-related pathways (p ≤ 0.05). Conclusions Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease. [ABSTRACT FROM AUTHOR]

In contrast to yeast and animal cells, plants contain two genes which is encoding to nucleolin proteins. A comprehensive protein sequence analysis revealed that the two RNA recognition motifs (RRM) domains are highly conserved and suggesting that the mechanisms and functions related to RNA contained RRMs are highly conserved in both plant nucleolin proteins sequences. Whereas Ribonucleoprotein 1 (RNP1) and Ribonucleoprotein 2 (RNP2) that are highly conserved sequences in RRMs from yeast and animal nucleolin, also my study confirmed that RNP1 and RNP2 sequences are conserved in plants. Nucleolin could use the same manner in the different species of plants and animals to achieve its function. Study of evolutionary relationships among nine different plant species revealed that monocots and dicots form two separated monophyletic group meaning that they composed of a collection of organisms, share a common evolutionary history and including the most recent common ancestor of all those organisms. [ABSTRACT FROM AUTHOR]

Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions. [ABSTRACT FROM AUTHOR]

Selective occlusion of tumor vasculature has proven to be an effective strategy for cancer therapy. Among vascular coagulation agents, the extracellular domain of coagulation-inducing protein tissue factor, truncated tissue factor (tTF), is the most widely used. Since the truncated protein exhibits no coagulation activity and is rapidly cleared in the circulation, free tTF cannot be used for cancer treatment on its own but must be combined with other moieties. We here developed a novel, tumor-specific tTF delivery system through coupling tTF with the DNA aptamer, AS1411, which selectively binds to nucleolin receptors overexpressing on the surface of tumor vascular endothelial cells and is specifically cytotoxic to target cells. Systemic administration of the tTF-AS1411 conjugates into tumor-bearing animals induced intravascular thrombosis solely in tumors, thus reducing tumor blood supply and inducing tumor necrosis without apparent side effects. This conjugate represents a uniquely attractive candidate for the clinical translation of vessel occlusion agent for cancer therapy. This study reported a novel, tumor-specific tTF delivery system through coupling tTF with the DNA aptamer, AS1411, which selectively binds to nucleolin receptors overexpressing on the surface of tumor vascular endothelial cells and is specifically cytotoxic to target cells. [Display omitted] [ABSTRACT FROM AUTHOR]