The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

Critical role of G3BP1 in bovine parainfluenza virus type 3 (BPIV3)-inhibition of stress granules formation and viral replication.

Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.

TRIM25 predominately associates with anti-viral stress granules.

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Biomarkers for prognosis of meningioma patients: A systematic review and meta-analysis.

Meningioma is the most common primary brain tumor and many studies have evaluated numerous biomarkers for their prognostic value, often with inconsistent results. Currently, no reliable biomarkers are available to predict the survival, recurrence, and progression of meningioma patients in clinical practice. This study aims to evaluate the prognostic value of immunohistochemistry-based (IHC) biomarkers of meningioma patients. A systematic literature search was conducted up to November 2023 on PubMed, CENTRAL, CINAHL Plus, and Scopus databases. Two authors independently reviewed the identified relevant studies, extracted data, and assessed the risk of bias of the studies included. Meta-analyses were performed with the hazard ratio (HR) and 95% confidence interval (CI) of overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). The risk of bias in the included studies was evaluated using the Quality in Prognosis Studies (QUIPS) tool. A total of 100 studies with 16,745 patients were included in this review. As the promising markers to predict OS of meningioma patients, Ki-67/MIB-1 (HR = 1.03, 95%CI 1.02 to 1.05) was identified to associate with poor prognosis of the patients. Overexpression of cyclin A (HR = 4.91, 95%CI 1.38 to 17.44), topoisomerase II α (TOP2A) (HR = 4.90, 95%CI 2.96 to 8.12), p53 (HR = 2.40, 95%CI 1.73 to 3.34), vascular endothelial growth factor (VEGF) (HR = 1.61, 95%CI 1.36 to 1.90), and Ki-67 (HR = 1.33, 95%CI 1.21 to 1.46), were identified also as unfavorable prognostic biomarkers for poor RFS of meningioma patients. Conversely, positive progesterone receptor (PR) and p21 staining were associated with longer RFS and are considered biomarkers of favorable prognosis of meningioma patients (HR = 0.60, 95% CI 0.41 to 0.88 and HR = 1.89, 95%CI 1.11 to 3.20). Additionally, high expression of Ki-67 was identified as a prognosis biomarker for poor PFS of meningioma patients (HR = 1.02, 95%CI 1.00 to 1.04). Although only in single studies, KPNA2, CDK6, Cox-2, MCM7 and PCNA are proposed as additional markers with high expression that are related with poor prognosis of meningioma patients. In conclusion, the results of the meta-analysis demonstrated that PR, cyclin A, TOP2A, p21, p53, VEGF and Ki-67 are either positively or negatively associated with survival of meningioma patients and might be useful biomarkers to assess the prognosis.

Expression and potential molecular mechanism of TOP2A in metastasis of non-small cell lung cancer.

DNA topoisomerase II alpha (TOP2A) expression, gene alterations, and enzyme activity have been studied in various malignant tumors. Abnormal elevation of TOP2A expression is considered to be related to the development of non-small cell lung cancer (NSCLC). However, its association with tumor metastasis and its mode of action remains unclear. Bioinformatics, real-time quantitative PCR, immunohistochemistry and immunoblotting were used to detect TOP2A expression in NSCLC tissues and cells. Cell migration and invasion assays as well as cytoskeletal staining were performed to analyze the effects of TOP2A on the motility, migration and invasion ability of NSCLC cells. Cell cycle and apoptosis assays were used to verify the effects of TOP2A on apoptosis as well as cycle distribution in NSCLC. TOP2A expression was considerably upregulated in NSCLC and significantly correlated with tumor metastasis and the occurrence of epithelial-mesenchymal transition (EMT) in NSCLC. Additionally, by interacting with the classical ligand Wnt3a, TOP2A may trigger the canonical Wnt signaling pathway in NSCLC. These observations suggest that TOP2A promotes EMT in NSCLC by activating the Wnt/ß-catenin signaling pathway and positively regulates malignant events in NSCLC, in addition to its significant association with tumor metastasis. TOP2A promotes the metastasis of NSCLC by stimulating the canonical Wnt signaling pathway and inducing EMT. This study further elucidates the mechanism of action of TOP2A, suggesting that it might be a potential therapeutic target for anti-metastatic therapy.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin ß1/FAK signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PURPOSE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin ß1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin ß1, FAK, ß-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin ß1, ß-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin ß1/FAK pathway. CONCLUSION: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin ß1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

A new G3BP1-GFP reporter system for assessing skin toxicity by real-time monitoring of stress granules in vitro.

The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.

Infection with SARS-CoV-2 can cause pancreatic impairment.

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (ß) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor ß. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Transcription-coupled DNA-protein crosslink repair by CSB and CRL4CSA-mediated degradation.

DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.

Knockdown of TOP2A suppresses IL-17 signaling pathway and alleviates the progression of ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa, with a gradually increasing incidence. Therefore, it is necessary to actively seek targets for the treatment of UC. METHODS: Common differentially expressed genes (DEGs) were screened from two microarray data sets related to UC. Protein-protein interaction network was constructed to find the hub genes. The UC mouse model and cell model were induced by dextran sulfate sodium (DSS). The pathological changes of colon tissue were observed by hematoxylin-eosin staining. Immunohistochemistry and immunofluorescence were performed to detect the expressions of Ki67 and Claudin-1. The performance of mice was observed by disease activity index (DAI). The effect of TOP2A on proliferation, inflammation, oxidative stress, and interleukin-17 (IL-17) signaling pathway in UC model was measured by cell counting kit-8, enzyme-linked immunosorbent assay, and western blot. RESULTS: Through bioinformatics analysis, 295 common DEGs were screened, and the hub gene TOP2A was selected. In UC model, there was obvious inflammatory cell infiltration in the colon and less goblet cells, while si-TOP2A lessened it. More Ki67 positive cells and less Claudin-1 positive cells were observed in UC model mice. Furthermore, knockdown of TOP2A increased the body weight and colon length of UC mice, while the DAI was decreased. Through in vivo and in vitro experiments, knockdown of TOP2A also inhibited inflammation and IL-17 signaling pathway, and promoted proliferation in DSS-induced NCM460 cells. CONCLUSION: Knockdown of TOP2A alleviated the progression of UC by suppressing inflammation and inhibited IL-17 signaling pathway.

Spectrum of ERCC6-Related Cockayne Syndrome (Type B): From Mild to Severe Forms.

(1) Background: Cockayne syndrome (CS) is an ultra-rare multisystem disorder, classically subdivided into three forms and characterized by a clinical spectrum without a clear genotype-phenotype correlation for both the two causative genes ERCC6 (CS type B) and ERCC8 (CS type A). We assessed this, presenting a series of patients with genetically confirmed CSB. (2) Materials and Methods: We retrospectively collected demographic, clinical, genetic, neuroimaging, and serum neurofilament light-chain (sNFL) data about CSB patients; diagnostic and severity scores were also determined. (3) Results: Data of eight ERCC6/CSB patients are presented. Four patients had CS I, three patients CS II, and one patient CS III. Various degrees of ataxia and spasticity were cardinal neurologic features, with variably combined systemic characteristics. Mean age at diagnosis was lower in the type II form, in which classic CS signs were more evident. Interestingly, sNFL determination appeared to reflect clinical classification. Two novel premature stop codon and one novel missense variants were identified. All CS I subjects harbored the p.Arg735Ter variant; the milder CS III subject carried the p.Leu764Ser missense change. (4) Conclusion: Our work confirms clinical variability also in the ERCC6/CSB type, where manifestations may range from severe involvement with prenatal or neonatal onset to normal psychomotor development followed by progressive ataxia. We propose, for the first time in CS, sNFL as a useful peripheral biomarker, with increased levels compared to currently available reference values and with the potential ability to reflect disease severity.

A new tandem repeat-enriched lncRNA XLOC_008672 promotes gastric carcinogenesis by regulating G3BP1 expression.

Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.

Exosomal miRNA-92a derived from cancer-associated fibroblasts promote invasion and metastasis in breast cancer by regulating G3BP2.

Cancer-associated Fibroblasts (CAFs) exert a tumor-promoting effect in various cancers, including breast cancer. CAFs secrete exosomes containing miRNA and proteins, influencing the tumor microenvironment. In this study, we identified CAF-derived exosomes that transport functional miR-92a from CAFs to tumor cells, thereby intensifying the aggressiveness of breast cancer. CAFs downregulate the expression of G3BP2 in breast cancer cells, and a significant elevation in miR-92a levels in CAF-derived exosomes was observed. Both in vitro and in vivo experiments demonstrate that miR-92a enhances breast cancer cell migration and invasion by directly targeting G3BP2, functioning as a tumor-promoting miRNA. We validated that the RNA-binding proteins SNRPA facilitate the transfer of CAF-derived exosomal miR-92a to breast cancer cells. The reduction of G3BP2 protein by CAF-derived exosomes releases TWIST1 into the nucleus, promoting epithelial-mesenchymal transition (EMT) and further exacerbating breast cancer progression. Moreover, CAF-derived exosomal miR-92a induces tumor invasion and metastasis in mice. Overall, our study reveals that CAF-derived exosomal miR-92a serves as a promoter in the migration and invasion of breast cancer cells by reducing G3BP2 and may represent a potential novel tumor marker for breast cancer.

Phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response.

Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.

The correlation study between TOP2A gene expression in circulating tumor cells and chemotherapeutic drug resistance of patients with breast cancer.

BACKGROUND: Patients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients. PATIENTS AND METHODS: A total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan-Meier survival curve. RESULTS: The patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression. CONCLUSIONS: High TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS.