Exosomal miR-130a-3p confers cisplatin resistance in esophageal cancer by regulating ferroptosis via the suppression of METTL14-mediated m6A RNA methylation of FSP1.

Exosomal microRNA (miRNA)s have been proven to affect recipient cell chemoresistance in several cancers. This research attempted to uncover the role of exosomal miRNA and the regulatory mechanism in cisplatin resistance of esophageal cancer (EC). Cisplatin-resistant EC cells (KYSE-150-CisR and TE-1-CisR) were established by the parental cells (KYSE-150 and TE-1) treated with a gradual increase of cisplatin concentration. Exosomes from both cisplatin-resistant EC cells and the parental one were obtained with ultracentrifugation (CisR-exo and CisS-exo), and identified by transmission electron microscopy and nanoparticle tracking analysis. The effect of CisR-exo on the cisplatin resistance of EC was assessed by in vitro (and in vivo functional experiments.Intracellular reactive oxygen species and iron were determined by the corresponding kits. The m6A dot blot assay and methylated RIP-qPCR was conducted to analyze the m6A modification level. Dual-luciferase reporter assay was performed to confirm the intermolecular interaction. Increased levels of miR-130a-3p were observed in both KYSE-150CisR and TE-1CisR cells, as well as their derived CisR-exos when compared with the parental cells and CisS-exos. Exosomal miR-130a-3p from cisplatin-resistant EC cells conferred cisplatin resistance to EC in vitro and in vivo, which might be mediated by the suppression of ferroptosis. Mechanically, KYSE-150CisR derived exosomal miR-130a-3p interacted with METTL14 to inhibit FSP1 (a ferroptosis suppressor) m6A modification of recipient cells. Overexpressing METTL14 restrained the cisplatin resistance disseminated by CisR-exos in KYSE-150 cells. Cisplatin-resistant EC cell-isolated exosomal miR-130a-3p suppressed the m6A modification of FSP1 to modulate ferroptosis, enhancing cisplatin resistance.

Han H ，Li Y ，Lin Z ，Ma X ，Huang W ，Lu C ，Ma R ，Han R ... -  《-》

Hypoxic BMSC-derived exosomal miR-210-3p promotes progression of triple-negative breast cancer cells via NFIX-Wnt/β-catenin signaling axis.

Bone marrow mesenchymal stem cells (BMSCs) are a crucial component of the tumor microenvironment (TME), with hypoxic conditions promoting their migration to tumors. Exosomes play a vital role in cell-to-cell communication within the TME. Hypoxic TME have a great impact on the release, uptake and biofunctions of exosomes. This study aims to elucidate the communication between BMSC-derived exosomal miRNA and triple-negative breast cancer (TNBC) in a hypoxic environment. Exosomes were isolated via ultracentrifugation and identified using scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA) and western blot. A range of bioinformatics approaches were used to screen exosomal miRNAs and the target mRNAs of miRNAs and predict the possible signaling pathways. Expression levels of genes and proteins were assessed by quantitative real-time PCR and western blot. Cell proliferation, apoptosis, migration and invasion were analyzed using CCK-8 assay, EDU assay, transwell migration, wound healing assay and invasion assay, respectively. Dual luciferase reporter gene assay was conducted to confirm the binding between miRNAs and the target mRNAs. The impact of hypoxic BMSC-derived exosomal miRNA on TNBC progression in vivo was evaluated using tumor xenograft nude mouse models. Furthermore, the impact of patients' serum exosomal miRNA on TNBC was implemented. Exosomes derived from hypoxic BMSCs promotes the proliferation, migration, invasion and epithelial-mesenchymal transition of TNBC and suppresses the apoptosis of TNBC. The expression of miR-210-3p in BMSC-derived exosomes is markedly elevated in hypoxic conditions. Exosome-mediated transfer of miR-210-3p from hypoxic BMSCs to TNBC targets NFIX and activates Wnt/β-Catenin signaling in TNBC. Deletion of miR-210-3p in hypoxic BMSC-derived exosomes attenuates TNBC in vivo. Additionally, human exosomal miR-210-3p from the serum of TNBC patients promotes TNBC progression. Moreover, we notably observed a marked downregulation of NFIX expression levels in cancerous tissues compared to paracancerous tissues. Hypoxic BMSC-derived exosomal miR-210-3p promotes TNBC progression via NFIX-Wnt/β-catenin signaling axis.

Wang M ，Zheng Y ，Hao Q ，Mao G ，Dai Z ，Zhai Z ，Lin S ，Liang B ，Kang H ，Ma X ... -  《Journal of Translational Medicine》

Decreased lncRNA HNF4A-AS1 facilitates resistance to sorafenib-induced ferroptosis of hepatocellular carcinoma by reprogramming lipid metabolism.

Background: Resistance to sorafenib remains a major challenge in the systemic therapy of liver cancer. However, the involvement of lipid metabolism-related lncRNAs in this process remains unclear. Methods: Different expression levels of lipid metabolism-related lncRNAs in HCC were compared by analysis of Gene Expression Omnibus and The Cancer Genome Atlas databases. The influence of HNF4A-AS1 on sorafenib response was evaluated through analysis of public biobanks, cell cytotoxicity and colony formation assays. The effect of HNF4A-AS1 on sorafenib-induced ferroptosis was measured using lipid peroxidation, glutathione, malondialdehyde, and ROS levels. Furthermore, bioinformatic analyses and lipidomic profiling were conducted to study HNF4A-AS1 involvement in lipid metabolic reprogramming. Mechanistic experiments, including the luciferase reporter assay, RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), and RNA remaining assays, were employed to uncover the downstream targets and regulatory mechanisms of HNF4A-AS1 in sorafenib resistance in HCC. Xenograft and organoid experiments were carried out to assess the impact of HNF4A-AS1 on sorafenib response. Results: Bioinformatics analysis revealed that HNF4A-AS1, a lipid metabolism-related lncRNA, is specifically high-expressed in the normal liver and associated with sorafenib resistance in HCC. We further confirmed that HNF4A-AS1 was downregulated in HCC cells and organoids that resistant to sorafenib. Moreover, both in vitro and in vivo studies demonstrated that HNF4A-AS1 overexpression reversed sorafenib resistance in HCC cells, which was further enhanced by polyunsaturated fatty acids (PUFA) supplementation. Mechanistically, HNF4A-AS1 interacted with METTL3, leading to m6A modification of DECR1 mRNA, which subsequently decreased DECR1 expression via YTHDF3-dependent mRNA degradation. Consequently, decreased HNF4A-AS1 levels caused DECR1 overexpression, leading to decreased intracellular PUFA content and promoting resistance to sorafenib-induced ferroptosis in HCC. Conclusions: Our results indicated the pivotal role of lipid metabolism-related and liver-specific HNF4A-AS1 in inhibiting sorafenib resistance by promoting ferroptosis and suggesting that HNF4A-AS1 might be a potential target for HCC.

Zhao Y ，Han S ，Zeng Z ，Zheng H ，Li Y ，Wang F ，Huang Y ，Zhao Y ，Zhuo W ，Lv G ，Wang H ，Zhao G ，Zhao E ，Hu Y ，Hu P ，Zhao G ... -  《Theranostics》

EGCG targeting STAT3 transcriptionally represses PLXNC1 to inhibit M2 polarization mediated by gastric cancer cell-derived exosomal miR-92b-5p.

M2-polarized tumor-associated macrophages (TAMs) predominate in tumor microenvironment (TME) and serve primary functions in tumor progression, including growth, angiogenesis, metastasis, immunosuppression, chemoresistance, and poor prognosis. The reversal of M2 polarization provides a new treatment strategy for cancer. Presently, the molecular mechanisms of M2 polarization have yet to be fully characterized, and there is a lack of effective therapeutic targets and drugs. Cancer cells initiate an immunosuppressive TME by recruiting macrophages and promoting M2 polarization through the secretion of inflammatory factors. Accordingly, blocking cancer cell-induced TAM M2 polarization may present a more effective strategy from the perspective of cancer cells. Hedyotis diffusa Willd (HDW) possesses immunomodulatory and antitumor properties, and is a precious and direct source of small molecule natural products with a dual function of inhibition of tumor growth and tumor cell-mediated M2 polarization. To identify a new target promoting gastric cancer (GC) cell growth and GC cell-mediated M2 polarization from mRNA profiles of GC cells treated with HDW injection (HDI) and to excavate a natural product from HDI that can regulate related mRNA and inhibit the aforementioned effects. RNA sequencing (RNA-seq) was used to analyze HDI-regulated differentially expressed mRNAs (HRmRNAs) in MKN45 cells. Weighted gene co-expression network analysis (WGCNA), univariate and multivariate Cox regression analysis, KM survival curves, and association analysis between HRmRNA and clinical characteristics/tumor infiltrating immune cells (TIICs) individually were utilized to screen out the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. shRNA lentiviral vectors were used for stably silencing, and transient overexpressing plasmids were constructed for overexpression. CCK8, EdU, colony formation, migration and invasion assays were used to validate the function of drugs and molecules in GC. HDI constituent analysis was performed using UHPLC-QE-MS. A network of HDI constituent-hub transcription factor (TF)-HRmRNA was constructed based on RNA-Seq, network pharmacology and TFs prediction. Exosome isolation and identification were performed using ultracentrifugation, NTA, TEM and western blot. Apoptosis and macrophage phenotypes were determined by flow cytometric analysis. Small RNA-Seq made exosomal miRNA identification. Small molecule interaction with targets were analyzed using molecular docking, SPR and CETSA. The direct relationship between transcription factors and promoters was verified using ChIP-QPCR and dual-luciferase reporter gene assay. A nude mice xenograft tumor model was established for vivo validation. HDI inhibited MKN45 cell proliferation, migration, invasion and promoted apoptosis. RNA-Seq identified 2583 HRmRNAs. PLXNC1 was screened out as the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. PLXNC1 promoted GC cell proliferation and facilitated TAMs M2 polarization by transferring GC cell-derived exosomal miR-92b-5p, inhibiting SOCS7-STAT3 interactions and subsequently activating STAT3 in macrophages. M2 TAMs induced by PLXNC1-mediated GC cell-derived exosomes promoted GC cell migration and invasion. PLXNC1 regulated exosomal miR-92b-5p through the MEK1/MSK1/CREB1 pathway. STAT3 could transcriptionally regulate PLXNC1 expression in GC cells. The network of HDI constituent-hub TF-HRmRNA showed epigallocatechin gallate (EGCG) from HDI targeted STAT3 to transcriptionally regulate PLXNC1 expression. EGCG as a natural product directly bound to STAT3 to diminish its nuclear localization, resulting in the transcriptional repression of PLXNC1 and the reversal of M2 polarization induced by PLXNC1-mediated GC cell-derived exosomes. PLXNC1 is a novel target exerting dual effects on GC cell proliferation and GC cell-mediated M2 polarization. EGCG derived from HDI inhibits GC cell proliferation and targets STAT3 to inhibit M2 polarization induced by PLXNC1-mediated exosomes derived from GC cells, which may be a multi-target therapeutic agent for GC cell proliferation and immune microenvironment.

Yi J ，Ye Z ，Xu H ，Zhang H ，Cao H ，Li X ，Wang T ，Dong C ，Du Y ，Dong S ，Zhou W ... -  《-》

Exosomes-Shuttled lncRNA SNHG7 by Bone Marrow Mesenchymal Stem Cells Alleviates Osteoarthritis Through Targeting miR-485-5p/FSP1 Axis-Mediated Chondrocytes Ferroptosis and Inflammation.

Osteoarthritis (OA), a degenerative joint disorder, is a major reason of disability in adults. Accumulating evidences have proved that bone marrow mesenchymal stem cells (BMSCs)-carried exosomes play a significant therapeutic effect on OA. However, the precise regulatory network remains unknown. OA and normal cartilage samples were acquired from patients, and chondrocytes were exposed to IL-1β to conduct a cellular OA model. Exosomes prepared from BMSCs were identified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Cell viability was determined with CCK-8 assay. Inflammatory injury was assessed by LDH and inflammatory factors (TNF-α and IL-6) using corresponding ELISA kits, respectively. Ferroptosis was evaluated by GSH, MDA and iron levels using corresponding kits, and ROS level with DCFH-DA. The expressions of genes/proteins were determined with RT-qPCR/western bolt. RNA immunoprecipitation and luciferase activity assay were conducted for testing the interactions of small nucleolar RNA host gene 7 (SNHG7)/ferroptosis suppressor protein 1 (FSP1) and miR-485-5p. The expressions of SNHG7 and FSP1 were both reduced in IL-1β-induced chondrocytes and OA cartilage tissues, and there was a positive correlation between them in clinical level. Moreover, SNHG7 was enriched in BMSCs-derived exosomes (BMSCs-Exos) and could be internalized by chondrocytes. Functional analysis illustrated that BMSCs-Exos administration repressed inflammatory injury, oxidative stress and ferroptosis in IL-1β-induced chondrocytes, while these changes were reinforced when SNHG7 was overexpressed in BMSCs-Exos. Notably, FSP1 silencing in chondrocytes abolished the beneficial effects mediated by exosomal SNHG7. Exosomal SNHG7 released from BMSCs inhibited inflammation and ferroptosis in IL-1β-induced chondrocytes through miR-485-5p/FSP1 axis. This work suggested that BMSCs-derived exosomal SNHG7 would be a prospective target for OA treatment.

Wang Y ，Hu K ，Liao C ，Han T ，Jiang F ，Gao Z ，Yan J ... -  《-》