Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Therapeutic targeting of nuclear export and import receptors in cancer and their potential in combination chemotherapy.

Systemic modalities are crucial in the management of disseminated malignancies and liquid tumours. However, patient responses and tolerability to treatment are generally poor and those that enter remission often return with refractory disease. Combination therapies provide a methodology to overcome chemoresistance mechanisms and address dose-limiting toxicities. A deeper understanding of tumorigenic processes at the molecular level has brought a targeted therapy approach to the forefront of cancer research, and novel cancer biomarkers are being identified at a rapid rate, with some showing potential therapeutic benefits. The Karyopherin superfamily of proteins is soluble receptors that mediate nucleocytoplasmic shuttling of proteins and RNAs, and recently, nuclear transport receptors have been recognized as novel anticancer targets. Inhibitors against nuclear export have been approved for clinical use against certain cancer types, whereas inhibitors against nuclear import are in preclinical stages of investigation. Mechanistically, targeting nucleocytoplasmic shuttling has shown to abrogate oncogenic signalling and restore tumour suppressor functions through nuclear sequestration of relevant proteins and mRNAs. Hence, nuclear transport inhibitors display broad spectrum anticancer activity and harbour potential to engage in synergistic interactions with a wide array of cytotoxic agents and other targeted agents. This review is focussed on the most researched nuclear transport receptors in the context of cancer, XPO1 and KPNB1, and highlights how inhibitors targeting these receptors can enhance the therapeutic efficacy of standard of care therapies and novel targeted agents in a combination therapy approach. Furthermore, an updated review on the therapeutic targeting of lesser characterized karyopherin proteins is provided and resistance to clinically approved nuclear export inhibitors is discussed.

Maize DDK1 encoding an Importin-4 ß protein is essential for seed development and grain filling by mediating nuclear exporting of eIF1A.

Nuclear-cytoplasmic trafficking is crucial for protein synthesis in eukaryotic cells due to the spatial separation of transcription and translation by the nuclear envelope. However, the mechanism underlying this process remains largely unknown in plants. In this study, we isolated a maize (Zea mays) mutant designated developmentally delayed kernel 1 (ddk1), which exhibits delayed seed development and slower filling. Ddk1 encodes a plant-specific protein known as Importin-4 ß, and its mutation results in reduced 80S monosomes and suppressed protein synthesis. Through our investigations, we found that DDK1 interacts with eIF1A proteins in vivo. However, in vitro experiments revealed that this interaction exhibits low affinity in the absence of RanGTP. Additionally, while the eIF1A protein primarily localizes to the cytoplasm in the wild-type, it remains significantly retained within the nuclei of ddk1 mutants. These observations suggest that DDK1 functions as an exportin and collaborates with RanGTP to facilitate the nuclear export of eIF1A, consequently regulating endosperm development at the translational level. Importantly, both DDK1 and eIF1A are conserved among various plant species, implying the preservation of this regulatory module across diverse plants.

RNA-binding protein hnRNPU regulates multiple myeloma resistance to selinexor.

Multiple myeloma (MM) is an incurable haematological cancer. Selinexor is the first-in-class selective inhibitor of nuclear export (SINE) and was newly approved for the treatment of MM. Until now, very few studies have investigated selinexor resistance in MM. Heterogeneous nuclear ribonucleoprotein U (hnRNPU) is an RNA-binding protein and a component of hnRNP complexes. Here we found that hnRNPU regulates MM sensitivity to selinexor. Cell apoptosis assays were performed to compare selinexor-induced cell death in control knockdown (CTR-KD) and hnRNPU knockdown (hnR-KD) MM cells. HnRNPU knockdown-induced nuclear protein retention was examined by proteomics array. HnRNPU-conferred mRNA translation regulation was evaluated by sucrose gradient assay, RNA electrophoresis mobility shift assay, and RNA pull-down assay. We found that hnR-KD MM cells were more sensitive to selinexor-induced cell death in vitro and in mouse model. MM patients who responded to selinexor had relatively low hnRNPU expression. In brief, hnRNPU comprehensively regulated MM sensitivity to selinexor by affecting the localization of LTV1 and NMD3, and mRNA translation of MDM2 and RAN, which were involved in XPO1-mediated nuclear export of ribosome subunits and tumor suppressors. Our discoveries indicate that hnRNPU might be a possible marker to categorize MM patients for the use of Selinexor.

Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis.

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments.

Nuclear transport proteins: structure, function, and disease relevance.

Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of many macromolecules within the nuclear or cytoplasmic compartments. In humans, approximately 60 proteins are involved in nuclear transport, including nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Many of these nuclear transport proteins play additional and essential roles in mitosis, biomolecular condensation, and gene transcription. Dysregulation of nuclear transport is linked to major human diseases such as cancer, neurodegenerative diseases, and viral infections. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing. This review summarizes approximately three decades of research data in this field but focuses on the structure and function of individual nuclear transport proteins from recent studies, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease. In-depth knowledge of this rapidly evolving field has the potential to bring new insights into fundamental biology, pathogenic mechanisms, and therapeutic approaches.

The relationship between prognosis of patients with traumatic brain injury and microRNA biogenesis proteins.

BACKGROUND: This study aims to investigate whether the expression levels of proteins involved in microRNA (miRNA) biogenesis vary in early- and late-stage traumatic brain injury (TBI) patients and to evaluate its effect on prognosis. METHODS: Dicer, Drosha, DiGeorge Syndrome Critical Region eight (DGCR8), Exportin5 (XPO5), and Argonaute2 (AGO2) levels were measured in the blood samples of severe TBI patients collected 4-6 h and 72 h after the trauma and compared with the control group. Prognostic follow-up of the patients was performed using the Glasgow Coma Scale score. RESULTS: There were no statistically significant changes in the expression of the miRNA biogenesis proteins Dicer, Drosha, DGCR8, XPO5, and AGO2 in patients with severe TBI. However, the expression of Dicer increased in the patients who improved from the severe TBI grade to the mild TBI grade, and the expression of AGO2 decreased in most of these patients. The Dicer expression profile was found to increase in patients discharged from the intensive care unit in a short time. CONCLUSION: MicroRNAs and their biogenesis proteins may guide prognostic and therapeutic decisions for patients with TBI in the future.

Lipid kinase PIP5K1A regulates let-7 microRNA biogenesis through interacting with nuclear export protein XPO5.

MicroRNAs (miRNAs) are small non-coding RNAs first discovered in Caenorhabditis elegans. The let-7 miRNA is highly conserved in sequence, biogenesis and function from C. elegans to humans. During miRNA biogenesis, XPO5-mediated nuclear export of pre-miRNAs is a rate-limiting step and, therefore, might be critical for the quantitative control of miRNA levels, yet little is known about how this is regulated. Here we show a novel role for lipid kinase PPK-1/PIP5K1A (phosphatidylinositol-4-phosphate 5-kinase) in regulating miRNA levels. We found that C. elegans PPK-1 functions in the lin-28/let-7 heterochronic pathway, which regulates the strict developmental timing of seam cells. In C. elegans and human cells, PPK-1/PIP5K1A regulates let-7 miRNA levels. We investigated the mechanism further in human cells and show that PIP5K1A interacts with nuclear export protein XPO5 in the nucleus to regulate mature miRNA levels by blocking the binding of XPO5 to pre-let-7 miRNA. Furthermore, we demonstrate that this role for PIP5K1A is kinase-independent. Our study uncovers the novel finding of a direct connection between PIP5K1A and miRNA biogenesis. Given that miRNAs are implicated in multiple diseases, including cancer, this new finding might lead to a novel therapeutic opportunity.

Adaptive laboratory evolution for acetic acid-tolerance matches sourdough challenges with yeast phenotypes.

Acetic acid tolerance of Saccharomyces cerevisiae is an important trait in sourdough fermentation processes, where the accumulation of acid by the growth of lactic acid bacteria reduces the yeast metabolic activity. In this work, we have carried out adaptive laboratory evolution (ALE) experiments in two sourdough isolates of S. cerevisiae exposed to acetic acid, or alternatively to acetic acid and myriocin, an inhibitor of sphingolipid biosynthesis that sped-up the evolutionary adaptation. Evolution approaches resulted in acetic tolerance, and surprisingly, increased lactic susceptibility. Four evolved clones, one from each parental strain and evolutionary scheme, were selected on the basis of their potential for CO2 production in sourdough conditions. Among them, two showed phenotypic instability characterized by strong lactic sensitivity after several rounds of growth under unstressed conditions, while two others, displayed increased constitutive acetic tolerance with no loss of growth in lactic medium. Genome sequencing and ploidy level analysis of all strains revealed aneuploidies, which could account for phenotypic heterogeneity. In addition, copy number variations (CNVs), affecting specially to genes involved in ion transport or flocculation, and single nucleotide polymorphisms (SNPs) were identified. Mutations in several genes, ARG82, KEX1, CTK1, SPT20, IRA2, ASG1 or GIS4, were confirmed as involved in acetic and/or lactic tolerance, and new determinants of these phenotypes, MSN5 and PSP2, identified.

Identification of the Teopod1, Teopod2, and Early Phase Change genes in maize.

Teopod1 (Tp1), Teopod2 (Tp2), and Early phase change (Epc) have profound effects on the timing of vegetative phase change in maize. Gain-of-function mutations in Tp1 and Tp2 delay all known phase-specific vegetative traits, whereas loss-of-function mutations in Epc accelerate vegetative phase change and cause shoot abortion in some genetic backgrounds. Here, we show that Tp1 and Tp2 likely represent cis-acting mutations that cause the overexpression of Zma-miR156j and Zma-miR156h, respectively. Epc is the maize ortholog of HASTY, an Arabidopsis gene that stabilizes miRNAs and promotes their intercellular movement. Consistent with its pleiotropic phenotype and epistatic interaction with Tp1 and Tp2, epc reduces the levels of miR156 and several other miRNAs.

Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3.

Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20's nuclear localization in RBM20-DCM patients.

Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.

NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure.

Investigation of the association of the RAN (rs14035) and XPO5 (rs11077) polymorphisms with venous thromboembolism.

INTRODUCTION: Venous thromboembolism (VTE) is the third most common hemostatic disease worldwide. Studies have reported a role for microRNA (miRNA) in the homeostasis and development of VTE. The ras-related nuclear protein (RAN) and exportin 5 (XPO5) genes are involved in miRNA biogenesis, as both regulate the transport of pre-miRNA from the nucleus to the cytoplasm. Therefore, the aim of the current study is to examine the association between RAN (rs14035) and XPO5 (rs11077) single nucleotide polymorphisms (SNPs) and VTE. METHODS: The study sample consisted of 300 subjects (150 patients and 150 age and sex matched controls). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system (T-ARMS) techniques were used to genotype rs14035 and rs11077, respectively. RESULTS: The results showed that there was a significant association between the XPO5 rs11077 and the risk of VTE (P < 0.05). Subjects with AC (OR: 2.08, CI:1.26-3.44) and CC (OR: 1.77, CI: 0.88-3.55) genotypes were at increased risk of the developing VTE. Regarding RAN gene, no association was found between rs14035 and VTE (P > 0.05). In addition, no associations were found between XPO5 rs11077 and RAN rs14035 genotypes with blood cell parameters (P > 0.05). As for the demographic characteristics, the results indicated a strong association between family history and body mass index (BMI) with the risk of VTE (P < 0.01). CONCLUSION: The XPO5 rs11077, BMI and family history might contribute to the development of VTE in Jordan.

Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis.

In eukaryotes, transcription factors are a crucial element in the regulation of gene expression, and nuclear translocation is the key to the function of transcription factors. Here, we show that the long intergenic noncoding RNA ARTA interacts with an importin ß-like protein, SAD2, through a long noncoding RNA-binding region embedded in the carboxyl terminal, and then it blocks the import of the transcription factor MYB7 into the nucleus. Abscisic acid (ABA)-induced ARTA expression can positively regulate ABI5 expression by fine-tuning MYB7 nuclear trafficking. Therefore, the mutation of arta represses ABI5 expression, resulting in desensitization to ABA, thereby reducing Arabidopsis drought tolerance. Our results demonstrate that lncRNA can hijack a nuclear trafficking receptor to modulate the nuclear import of a transcription factor during plant responses to environmental stimuli.

Nuclear export of BATF2 enhances colorectal cancer proliferation through binding to CRM1.

BACKGROUND: During the tumourigenesis and development of colorectal cancer (CRC), the inactivation of tumour suppressor genes is closely involved, although detailed molecular mechanisms remain elusive. Accumulating studies, including ours, have demonstrated that basic leucine zipper transcription factor ATF (activating transcription factor)-like 2 (BATF2) is a capable tumour suppressor that localises in the nucleus. However, its different subcellular localisation, potential functions and underlying mechanisms are unclear. METHODS: The translocation of BATF2 and its clinical relevance were detected using CRC samples, cell lines and xenograft nude mice. Candidate BATF2-binding proteins were screened using co-immunoprecipitation, quantitative label-free liquid chromatography-tandem mass spectrometry proteomic analysis, Western blotting and immunofluorescence. Recombinant plasmids, point mutations and siRNAs were applied to clarify the binding sites between BATF2 and chromosome region maintenance 1 (CRM1). RESULTS: The present study found that BATF2 was mainly localised in the cytoplasm, rather than nucleus, of CRC cells in vitro and in vivo, while cytoplasmic BATF2 expression was inversely correlated with the prognosis of CRC patients. Furthermore, we identified the nuclear export and subsequent ubiquitin-mediated degradation of BATF2 in CRC cells. Mechanistically, a functional nuclear export sequence (any amino acid) was characterised in BATF2 protein, through which BATF2 bound to CRM1 and translocated out of nucleus, ultimately enhancing CRC growth via inducing activator protein 1 (AP-1)/cyclin D1/phosphorylated retinoblastoma protein (pRb) signalling pathway. Additionally, nuclear export of BATF2 can be retarded by the mutation of NES in BATF2 or the knockdown of CRM1, whereas CRM1 expression was negatively associated with nuclear BATF2 expression and the prognosis of CRC patients. CONCLUSION: These findings revealed the biological effects and underlying mechanisms of cytoplasmic localisation of BATF2. Furthermore, suppressing nuclear export of BATF2 via mutating its NES region or inhibiting CRM1 expression may serve as a promising therapeutic strategy against CRC.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Identification of crucial genes involved in thyroid cancer development.

BACKGROUND: A malignancy of the endocrine system, one of the most common types, is thyroid cancer. It is proven that children who receive radiation treatment for leukemia or lymphoma are at a heightened risk of thyroid cancer due to low-dose radiation exposure throughout childhood. Several factors can increase the risk of thyroid cancer (ThyCa), such as chromosomal and genetic mutations, iodine intake, TSH levels, autoimmune thyroid disorders, estrogen, obesity, lifestyle changes, and environmental contaminants. OBJECTIVES: The study aimed to identify a specific gene as an essential candidate for thyroid cancer progression. We might be able to focus on developing a better understanding of how thyroid cancer is inherited. METHODS: The review article uses electronic databases such as PubMed, Google Scholar, Ovid MEDLINE, Embase, and Cochrane Central. The most frequently associated genes with thyroid cancer found on PubMed were BAX, XRCC1, XRCC3, XPO5, IL-10, BRAF, RET, and K-RAS. To perform an electronic literature search, genes derived from DisGeNET: a database of gene-disease associations, including PRKAR1A, BRAF, RET, NRAS, and KRAS, are used. CONCLUSION: Examining the genetics of thyroid cancer explicitly emphasizes the primary genes associated with the pathophysiology of young and older people with thyroid cancer. Developing such gene investigations at the beginning of the thyroid cancer development process can identify better outcomes and the most aggressive thyroid cancers.

Interference with XPO1 Suppresses the Stemness and Radioresistance of CD44 Positive Cervical Cancer Cells via Binding with Rad21.

OBJECTIVE: Cancer stem cells (CSCs) are responsible for cervical cancer progression and decreased radiosensitivity of tumor cells. The present work is meant to illuminate the impacts of exportin 1 (XPO1) on the aggressive behaviors and radiosensitivity of cervical cancer stemness cells and make a deeper inquiry into its regulatory mechanism despite that XPO1 has been supported to elicit significant activities on multiple malignancies. METHODS: XPO1 and Rad21 expression in HeLa (CD44+) cells was tested by RT-qPCR and western blot. Cell viability was estimated via CCK-8 assay. Cell stemness was examined via sphere formation assay and western blot. Following radiation treatment, cell proliferation was judged by CCK-8 assay, western blot as well as EdU staining whereas TUNEL assay, RT-qPCR and western blot analysis appraised cell apoptosis. Cell radiosensitivity was assessed through clonogenic survival assay. The levels of DNA damage markers were tested by western blot and related kits. STRING database and Co-IP assay respectively predicted and testified the binding of XPO1 with Rad21. RT-qPCR and western blot also examined the expression of XPO1 cargoes. RESULTS: The experimental data corroborated that XPO1 and Rad21 were overexpressed in cervical cancer tissues and cells. XPO1 inhibitor KPT-330 impeded the stemness while elevated the radiosensitivity of HeLa (CD44+) cells. XPO1 bond to Rad21 and positively modulated Rad21 expression. Moreover, Rad21 elevation reversed the impacts of KPT-330 on the behaviors of cervical cancer stemness cells. CONCLUSION: To sum up, XPO1 might impact the aggressive behavior and radioresistance of cervical cancer stemness cells through binding with Rad21.

Differential impact of exportin-1-mediated nuclear export of RNAs on the RNA content of extracellular vesicle subpopulations.

Extracellular vesicles (EVs) are membrane-enclosed subcellular structures released by all cell types. EVs have important roles in both cellular homeostasis and intercellular communication. Recent progress in the field revealed substantial heterogeneity of EVs even within the size-based EV categories. Here we addressed the question whether the exportin-1 (XPO1)-mediated nuclear export of RNAs contributed to the EV heterogeneity. Size-based populations were separated from the conditioned media of three cell lines (U937, THP-1 and 5/4E8) in steady-state condition. The effects of activation and leptomycin B treatment (to inhibit the XPO1-mediated nuclear export of RNAs) were also tested in the case of the two monocytic cell lines. Agilent Pico and Small chips were used to characterize RNAs, fragment analysis was performed, and EV-associated miRNAs were tested by Taqman assays. As expected, we found the highest small RNA/total RNA ratio and the lowest rRNA/total RNA proportion in small EVs (~ 50-150 nm). Profiles of the small RNAs within different size-based EV categories significantly differed based on the activation status of the EV releasing cells. Leptomycin B had a differential inhibition on the tested small RNAs in EVs, even within the same EV size category. A similar heterogeneity of the EV miRNA content was observed upon cellular activation and nuclear export inhibition. Here we complement the already existing knowledge on EV heterogeneity by providing evidence that the RNA cargo varies depending on the EV size-based category, the releasing cell type, the functional status of the releasing cells and the exportin-1-mediated nuclear export of RNAs.