The used primers, siRNAs, and probes

Long non-coding RNAs (lncRNAs) constitute a critical class of regulatory molecules involved in cancer biology and play pivotal roles in tumor initiation and progression. Nevertheless, the biological functions of many newly identified lncRNAs in non-small cell lung cancer (NSCLC), as well as their potential therapeutic relevance, remain insufficiently characterized. In this study, high-throughput sequencing analysis of paired NSCLC tumor tissues and adjacent non-tumorous samples revealed that LINC00973 is significantly upregulated in tumor specimens. Moreover, elevated LINC00973 expression was found to be closely associated with poor clinical outcomes in patients with NSCLC. Functional assays showed that LINC00973 knockdown inhibits NSCLC cell proliferation, migration, and invasion while inducing apoptosis, whereas overexpression produces opposite effects. These observations were confirmed in vivo, where LINC00973 depletion markedly suppressed tumor growth and metastasis. Mechanistically, LINC00973 interacts with and stabilizes deltex E3 ubiquitin ligase 3L (DTX3L), preventing its ubiquitination-mediated degradation and activating the AKT signaling pathway. Therapeutically, RGD-modified exosome-mediated delivery of LINC00973 siRNA significantly inhibited NSCLC progression in mouse models. Moreover, a synthetic biology-based strategy enabling hepatic production of exosomes carrying LINC00973-targeting siRNA achieved robust anti-tumor effects. Together, these findings establish LINC00973 as an oncogenic lncRNA that promotes NSCLC progression via DTX3L stabilization and highlight LINC00973 as a promising therapeutic target.

RGD-functionalized Ce6-loaded neutrophil nanovesicles (RNC) enable targeted photoimmunotherapy for CRC. RNC triggers ROS/ICD, converts cold tumors to hot, and synergizes with anti-PD-1 to suppress primary and distant tumors.

Introduction: Mesenchymal stem cells (MSCs) play a significant role in shaping and regulating microenvironments. The factors influencing the glycolytic metabolic reprogramming of MSCs remain unknown. Methods: MSCs were isolated from human umbilical cords and identified using flow cytometry. Glycolysis-related gene expression, glucose consumption, and ATP and lactate production were measured in MSCs with HIF1A-AS3 overexpression or knockdown. The roles of HIF1A-AS3 in the glycolytic pathway and its downstream network were revealed by high-throughput sequencing. Results: HIF1A-AS3 enhanced the glycolytic level of MSCs by increasing glucose consumption, lactate production, ATP levels, and expression of key glycolytic enzymes (GLUT1, HK2, and PKM2). The downstream signaling pathway of HIF1A-AS3 might be associated with glycosylationrelated diseases. Discussion: It was demonstrated that HIF1A-AS3, a hypoxia-induced long non‑coding RNA, acted as a critical regulator of glycolytic reprogramming in MSCs. Additionally, HIF1A-AS3 upregulated a distinct subset of interferon‑stimulated genes, suggesting a potential link between metabolic reprogramming and immunomodulation in MSCs. Further mechanistic studies are warranted to elucidate how HIF1A-AS3 regulates these processes. Conclusion: The study reveals that HIF1A-AS3 is a regulator of the metabolic reprogramming of glycolysis in MSCs. These findings provide new insights into understanding the metabolic regulatory mechanism of MSCs.

ABSTRACT Extracellular vesicles (EVs) from immune cells represent a novel drug delivery system with inherent anti‐tumor properties. To remodel the immunosuppressive tumor microenvironment and mediate multimodal therapy, we produced neutrophil nanovesicles at high yield and loaded them with drug (doxorubicin, DOX), near‐infrared region II fluorescent dye (FD1080), and PD‐1 inhibitor (TFA) through a simple extrusion method and further modified them with DSPE‐PEG2000‐cRGD to enhance their tumor‐targeting ability. The RGD‐NNV@FD1080&DOX/TFA exhibited excellent photothermal effect and efficiently suppressed tumor progression in mouse xenograft tumor, PDX, and lung metastasis models. Upon laser irradiation, the RGD‐NNV@FD1080&DOX/TFA elicited the activation of anti‐tumor immunity with increased infiltration of mature dendritic cells (DCs), CD8 + T cells, and decreased infiltration of regulatory T cells (Tregs) in tumors. Single‐cell RNA sequencing revealed that the combination therapy increased the subtype of cytotoxic and memory T cells while decreasing that of exhausted T cells, and re‐polarized M2 TAMs and N2 TANs. The re‐challenge model further confirmed that RGD‐NNV@FD1080&DOX/TFA exerted effective long‐term immune memory in mouse models and displayed excellent biosafety in vivo. Overall, we designed a simple and potentially clinically applicable nanovesicle‐based nanomedicine delivery system that exhibits a highly efficient anti‐tumor effect by remodeling the tumor immune microenvironment and activating anti‐tumor immunity.

MS for detecting peptide translation

Neutrophils, serving as crucial innate immune cells, exert anti-tumor effects through cytotoxic mediators, antibody-dependent responses, and coordination of immune networks. They also release exosomes that carry bioactive molecules such as microRNAs (miRNAs). Our previous work identified neutrophil-derived exosomes (N-Exo) as contributors to their anti-tumor activity, but the underlying mechanisms remain unclear. Functional experiments integrating high-throughput sequencing and validation assays were performed to screen and identify key anti-tumor miRNAs in N-Exo. An in vivo subcutaneous xenograft mouse tumor model assessed the therapeutic effects of N-Exo-delivered miR-101-3p on tumor growth. Bioinformatics analysis combined with experimental validation, including dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP), elucidated the mechanism by which the key miRNA suppresses tumorigenesis through targeting specific genes and signaling pathways. Recombinant interleukin-36 gamma (rmIL-36γ) was used to stimulate neutrophils, and functional assays were performed to evaluate its effect. Hsa-miR-101-3p was enriched in N-Exo. N-Exo-delivered miR-101-3p directly targets MCL1 to suppress its expression and indirectly inhibits MCL1 transcription via regulation of the EZH2/c-Myc axis, collectively promoting apoptosis in gastric cancer (GC) cells. Furthermore, rmIL-36γ priming upregulated miR-101-3p expression in neutrophils and enhanced their anti-tumor effects. We demonstrate that N-Exo exerts tumor-suppressive effects by delivering miR-101-3p, which dually targets and suppresses MCL1 expression. Moreover, rmIL-36γ treatment enhances both miR-101-3p abundance and anti-tumor efficacy in neutrophils. These findings highlight the N-Exo/miR-101-3p/MCL1 axis as a therapeutic target and support cytokine priming as a strategy to enhance neutrophil-based cancer therapy.

Exosomes mediate cellular communications and have a profound impact on cancer progression. N2 neutrophils, which are polarized by factors from cancers, extensively infiltrate into tumor tissues and promote cancer progression via distinct mechanisms. However, the role and underlying mechanism of exosomes derived from N2 neutrophils (N2-EXO) in cancer remain to be investigated. Herein, we reported that N2-EXO enhanced the proliferation and metastasis of gastric cancer (GC) cells by promoting their stemness. In addition, miR-223-3p and miR-425-5p, which were highly expressed in N2-EXO from GC patients, promoted cancer metastasis and reduced cancer sensitivity to oxaliplatin. The cancer-promoting effect of N2-EXO was abolished by the addition of miRNA inhibitor both in vitro and in vivo. Mechanically, miR-223-3p and miR-425-5p directly targeted FOXO3 and PTEN genes, respectively, which synergistically promoted GC progression by regulating PI3K/AKT signaling pathway. Taken together, our results reveal a novel mechanism by which N2-EXO promotes GC progression, providing new insights into the function of exosomes from N2 neutrophils in cancer.

Peroxiredoxin 2 (PRDX2) is an antioxidant enzyme that has been reported to be overexpressed in various cancers. However, the role of PRDX2 in gastric cancer progression and its underlying mechanism remains unclear. Herein, we revealed the function of PRDX2 in gastric cancer progression and explored its molecule mechanism. We identified that PRDX2 was upregulated and associated with poor prognosis in gastric cancer. The knockdown of PRDX2 inhibited the proliferation, migration and invasion of gastric cancer cells in vitro and suppressed tumor growth in vivo. Mechanistically, PRDX2 interacted with PKM2 (pyruvate kinase isozyme type M2) and protected PKM2 from ubiquitination and degradation, which enhanced glycolysis in gastric cancer cells. The interaction between PRDX2 and PKM2 also enhanced the binding affinity between PKM2 and importin α5, which induced PKM2 nuclear translocation and activated STAT3 signaling pathway. In addition, STAT3 (signal transducer and activator of transcription 3) was identified to bind to PRDX2 gene promoter and upregulate PRDX2 expression, which forms a positive regulatory feedback loop in gastric cancer cells. The present study unravels the biological role of PRDX2 in cancer progression and illustrates the underlying molecular mechanism, which may provide a potential therapeutic target for gastric cancer.

Wound healing is a highly coordinated physiological process, which is essential for restoring the structural and functional integrity of damaged tissues. The present review explores the multifaceted roles of epigenetic modifications in wound healing and their potential as therapeutic targets. Epigenetic mechanisms, including DNA methylation, histone modifications, regulation by non‑coding RNAs (ncRNAs) and RNA methylation, influence the speed and quality of wound repair by regulating gene expression, cell function and intercellular signaling. During the hemostasis phase, DNA methylation of genes such as platelet endothelial aggregation receptor 1 can impact platelet function, while histone methylation and acetylation serve critical roles in modulating inflammation and fibroblast activation. ncRNAs, such as microRNAs and long ncRNAs, regulate cell proliferation, collagen deposition and scar formation. N6‑methyladenosine modifications, a type of RNA methylation, impact autophagy and fibrosis through their interaction with YTH domain family proteins. Key epigenetic regulators influence wound healing outcomes, providing valuable insights for the development of novel therapeutic strategies. However, challenges remain in translating these findings into clinical applications due to the complexity of epigenetic networks and the need for precise regulatory tools. Future research should focus on elucidating the cell‑specific and spatiotemporal regulatory mechanisms of epigenetic modifications in wound healing, and exploring their potential as therapeutic targets for reducing scar formation and preventing chronic wounds.

Exosomes are essential mediators of cellular communication and plays important roles in cancer. In addition to exosomes from tumor cells, the recent studies suggest that exosomes from tumor microenvironment (TME) cells are also critically involved in tumor progression. Tumor-associated neutrophils (TANs) represent a major component of TME cells and have active roles in tumor progression, while the function of their derived exosomes has not been well understood thus far. In this study, we discovered that exosomes from N2 TANs promoted the resistance of gastric cancer to chemotherapy by suppressing ferroptosis. Exosomes from N2 TANs were enriched in miR-9-3p, which could be transferred to gastric cancer cells to inhibit ACSL4 expression. Exosomes from miR-9-3p depleted N2 TANs showed a decreased effect on suppressing erastin-induced ferroptosis. Exosomes derived from N2 TANs antagonized the ferroptosis-promoting effects of ACSL4 overexpression in gastric cancer cells. Additionally, we found that exosomes from N2 TANs protected gastric cancer cells from ferroptosis induced by oxaliplatin, a commonly used drug for gastric cancer therapy, which could be rescued by targeted inhibition of miR-9-3p both in vitro and in vivo. The upregulation of miR-9-3p in N2 TANs was dependent on the activation of NF-κB pathway by gastric cancer cells-derived exosomes. Moreover, we revealed that the expression level of miR-9-3p was higher in human gastric cancer tissues than normal tissues and was negativity associated with patient survival. Conclusively, we reported a previous unclarified role of N2 TANs in chemoresistance via miR-9-3p-enriched exosomes-mediated downregulation of ASCL4 and the consequent suppression of ferroptosis, which may provide a new biomarker and therapeutic target for gastric cancer.

Chemotherapy resistance is a major cause of poor prognosis in gastric cancer patients and tumor microenvironment plays a critical role in conferring chemotherapy resistance. As a dominant source of tumor stromal cells, mesenchymal stem cells (MSCs) exert pro-oncogenic activities when reprogrammed to a cancer-associated fibroblast (CAF) phenotype. The precise mechanisms for MSC reprogramming and their subsequent role in chemotherapy resistance have not been fully understood. Herein, we reported that HIF1A-AS3, a lncRNA that was highly expressed in tumor-promoting MSCs, was upregulated in tumor tissues and serum of gastric cancer patients and associated with poor prognosis. The upregulation of HIF1A-AS3 reprogramed MSCs to acquire the CAF phenotype, which consequently enhanced the resistance of gastric cancer cells to oxaliplatin. Mechanistically, hypoxia related transcription factor HIF-1α induced high expression of HIF1A-AS3 in MSCs. Then, HIF1A-AS3 competitively sponged miR-142-3p and miR-24-3p, leading to the upregulation of PROX1 (prospero-related homeobox protein 1) gene expression. This further promoted the nuclear translocation of β-catenin and the activation of β-catenin signaling pathway in MSCs, which critically regulated their transition to CAFs. Finally, targeted inhibition of HIF1A-AS3 in hypoxia-MSCs through exosome-mediated siRNA delivery significantly suppressed gastric cancer growth and improved chemosensitivity in mouse tumor models. Conclusively, hypoxia-induced HIF1A-AS3 upregulation reprograms MSCs to CAFs through the miR-142-3p/miR-24-3p/PROX1/β-catenin axis, thereby promoting chemotherapy resistance in gastric cancer, which uncovers a new molecular mechanism for MSCs transition to CAFs in gastric cancer and provides a new target for the diagnosis and targeted therapy of gastric cancer.

M2-polarized tumor-associated macrophages (M2 TAMs) promote cancer progression. Exosomes mediate cellular communication in the tumor microenvironment (TME). However, the roles of exosomes from M2 TAMs in gastric cancer progression are unclear. Herein, it is reported that M2 TAMs-derived exosomes induced aerobic glycolysis in gastric cancer cells and enhanced their proliferation, metastasis, and chemoresistance in a glycolysis-dependent manner. It is identified that MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is enriched in M2 TAM exosomes and confirmed that MALAT1 transfer from M2 TAMs to gastric cancer cells via exosomes mediates this effect. Mechanistically, MALAT1 interacted with the δ-catenin protein and suppressed its ubiquitination and degradation by β-TRCP. In addition, MALAT1 upregulated HIF-1α expression by acting as a sponge for miR-217-5p. The activation of β-catenin and HIF-1α signaling pathways by M2 TAM exosomes collectively led to enhanced aerobic glycolysis in gastric cancer cells. Finally, a dual-targeted inhibition of MALAT1 in both gastric cancer cells and macrophages by exosome-mediated delivery of siRNA remarkably suppressed gastric cancer growth and improved chemosensitivity in mouse tumor models. Taken together, these results suggest that M2 TAMs-derived exosomes promote gastric cancer progression via MALAT1-mediated regulation of glycolysis. The findings offer a potential target for gastric cancer therapy.

Tumor cells remodel the phenotype and function of tumor microenvironment (TME) cells to favor tumor progression. Previous studies have shown that neutrophils in TME are polarized to N2 tumor-associated neutrophils (TANs) by tumor derived factors, thus promoting tumor growth and metastasis, angiogenesis, therapy resistance, and immunosuppression. Exosomes act as critical intercellular messengers in human health and diseases including cancer. So far, the biological roles of exosomes from N2 TANs in gastric cancer have not been well characterized. Herein, we represented the first report that exosomes from N2 TANs promoted gastric cancer metastasis in vitro and in vivo. We found that exosomes from N2 TANs transferred miR-4745-5p/3911 to gastric cancer cells to downregulate SLIT2 (slit guidance ligand 2) gene expression. Adenovirus-mediated overexpression of SLIT2 reversed the promotion of gastric cancer metastasis by N2 TANs derived exosomes. We further revealed that gastric cancer cells induced glucose metabolic reprogramming in neutrophils through exosomal HMGB1 (high mobility group protein B1)/NF-κB pathway, which mediated neutrophil N2 polarization and miR-4745-5p/3911 upregulation. We further employed ddPCR (droplet digital PCR) to detect the expression of miR-4745-5p/3911 in N2 TANs exosomes from human serum samples and found their increased levels in gastric cancer patients compared to healthy controls and benign gastric disease patients. Conclusively, our results indicate that N2 TANs facilitate cancer metastasis via regulation of SLIT2 in gastric cancer cells by exosomal miR-4745-5p/3911, which provides a new insight into the roles of TME cells derived exosomes in gastric cancer metastasis and offers a potential biomarker for gastric cancer diagnosis.

Circular RNAs are a class of noncoding RNAs with covalently linked 5 ' and 3 ' ends that arise from backsplicing events. The absence of a 5 ' cap and a 3 ' poly(A) tail makes circular RNAs relatively more stable than their linear counterparts. They are evolutionary conserved and tissue-specific, and some show disease-specific expression patterns. Although their biological functions remain largely unknown, circular RNAs have been shown to play regulatory roles by acting as microRNA sponges, regulators of RNA-binding proteins, alternative splicing, and parental gene expression, and they could even encode proteins. Over the past few decades, circular RNAs have attracted wide attention in oncology owing to their implications in various tumors. Many circular RNAs have been characterized as key players in gastrointestinal cancers and influence cancer growth, progression, metastasis, and therapeutic resistance. Accumulating evidence reveals that their unique characteristics, coupled with their critical roles in tumorigenesis, make circular RNAs promising non-invasive clinical biomarkers for gastrointestinal cancers. In the present review, we summarized the biological roles of the emerging circular RNAs and their potential as biomarkers and therapeutic targets, which may help better understand their clinical significance in the management of gastrointestinal cancers. 1 image

Compelling evidence has identified circRNAs as crucial regulators in initiation and progression of various cancers, including gastric cancer (GC). However, the function and regulatory mechanisms of circRNAs in GC remain largely unknown. In this study, attention is paid to a novel circular RNA circ1811, which exerts significant downregulated expression in GC tissues compared with adjacent non-cancerous tissues. The expression of circ1811 in GC tumor tissues is negatively correlated with the extent of lymphatic metastasis in GC patients. Overexpression of circ1811 inhibited GC cell proliferation, migration and invasion while promoting apoptosis, whereas knockdown of circ1811 led to the opposite effects. AGO2 RIP and dual luciferase reporter assays indicated that circ1811 directly sponges miR-632 to upregulate the expression of DAPK1. Collectively, circ1811 acts as a tumor-suppressor for GC progression by regulating the miR-632/DAPK1 axis. Our findings suggest the potential of circ1811 as ideal biomarker and therapeutic target for GC.

Liquid biopsy is a minimally invasive method that uses biofluid samples instead of tissue samples for cancer diagnosis. Exosomes are small extracellular vesicles secreted by donor cells and act as mediators of intercellular communication in human health and disease. Due to their important roles, exosomes have been considered as promising biomarkers for liquid biopsy. However, traditional methods for exosome isolation and cargo detection methods are time-consuming and inefficient, limiting their practical application. In the past decades, many new strategies, such as microfluidic chips, nanowire arrays and electrochemical biosensors, have been proposed to achieve rapid, accurate and high-throughput detection and analysis of exosomes. In this review, we discussed about the new advance in exosome-based liquid biopsy technology, including isolation, enrichment, cargo detection and analysis approaches. The comparison of currently available methods is also included. Finally, we summarized the advantages and limitations of the present strategies and further gave a perspective to their future translational use.

The interaction between tumor cells and stromal cells within the tumor microenvironment plays a critical role in cancer progression. Mesenchymal stem cells (MSCs) are important tumor stromal cells that exhibit pro-oncogenic activities when reprogrammed by the tumor. However, the precise mechanisms underlying MSC reprogramming in gastric cancer remain not well understood. QRT-PCR, western blot, and immunohistochemistry were used to examine gene and protein expression levels. In vitro and in vivo experiments were conducted to assess the biological functions of gastric cancer cells. RNA-sequencing, RNA immunoprecipitation (RIP), and meRIP assays were performed to investigate underlying molecular mechanisms. We found a significant increase in the expression and N6-methyladenosine (m6A) modification levels of colony-stimulating factor 2 (CSF2) in gastric cancer MSCs. CSF2 gene overexpression induced the reprogramming of normal MSCs into cancer-promoting MSCs, thereby enhancing the proliferation, migration, and drug resistance of gastric cancer cells through the secretion of various pro-inflammatory factors. Additionally, we demonstrated that the m6A reader IGF2BP2 bound to and stabilized CSF2 mRNA in gastric cancer MSCs. Notably, overexpression of IGF2BP2 mimicked the effect of CSF2 on MSCs, promoting gastric cancer progression. Finally, we unveiled that CSF2 induced the ubiquitination of Notch1 to reprogram MSCs. Our study highlights a critical role of IGF2BP2-mediated m6A modification of CSF2 in reprogramming MSCs, which presents a promising therapeutic target for gastric cancer.

tRNA-derived fragments (tRFs) are an emerging category of small non-coding RNAs that are generated from cleavage of mature tRNAs or tRNA precursors. The advance in high-throughput sequencing has contributed to the identification of increasing number of tRFs with critical functions in distinct physiological and pathophysiological processes. tRFs can regulate cell viability, differentiation, and homeostasis through multiple mechanisms and are thus considered as critical regulators of human diseases including cancer. In addition, increasing evidence suggest the extracellular tRFs may be utilized as promising diagnostic and prognostic biomarkers for cancer liquid biopsy. In this review, we focus on the biogenesis, classification and modification of tRFs, and summarize the multifaceted functions of tRFs with an emphasis on the current research status and perspectives of tRFs in cancer.

Introduction: Resident mesenchymal stem cells (MSCs) in the tumor microenvironment play an important role in tumor progression. Up to now, the mechanism of resident MSCs promoting gastric cancer cell migration remains unclear. Methods: We tested the migration ability of gastric cancer cells by transwell assays in this study. The inflammatory factors secreted by MSCs were detected by Luminex and ELISA. The activation of NF-κB signaling was detected by western blot. The exosomes derived from MSCs were isolated and identified by transmission electron microscope, nano-sight and western blot. The expression of miR-374a-5p was confirmed by qRT-PCR and its downstream target HAPLN1 by luciferase reporter assay. The expression of adhesion molecules of gastric cancer cells was detected by flow cytometry. Results: MiR-374a-5p could regulate the expression of inflammatory factors by activating NF-κB signaling. The increase of MCP-1 and the decrease of IFN-γ promoted the migration of gastric cancer cells. The miR-374a-5p in MSCs could be encapsulated and delivered to gastric cancer cells by exosomes derived from MSCs. Exogenous miR-374a-5p up-regulated the expression of adhesion molecules in gastric cancer cells by targeting HAPLN1. And miR-374a-5p-enriched exosomes also promoted the migration of gastric cancer cells. Conclusion: MiR-374a-5p promoted gastric cancer metastasis, and resident MSCs in the gastric cancer microenvironment played a major role in the regulation of gastric cancer metastasis. The study will provide new ideas and potential targets for the prevention and treatment of gastric cancer metastasis.