Dataset Information

MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway.

ABSTRACT: Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

SUBMITTER: Xu C

PROVIDER: S-EPMC10313823 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway.

Xu Chao C Wang Pan P Guo Haiyan H Shao Chuan C Liao Bin B Gong Sheng S Zhou Yanghao Y Yang Bingjie B Jiang Haotian H Zhang Gang G Wu Nan N Wu Nan N

Cell death discovery 20230630 1

Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatme ...[more]

PMID: 37391426

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Project description:Background: Glioma-associated macrophages (GAMs) are one of the most important cellular components in the tumor immune microenvironment (TIME). GAMs display a mostly M2-like phenotype with anti-inflammatory features, which is closely related to the malignancy and progression of cancers. Extracellular vesicles derived from GAMs (GAM-EVs), which are important components of the TIME, contain a variety of microRNAs (miRNAs). These miRNAs play a crucial role in communication between tumor cells and GAMs. In this study, we aimed to explore the effect of GAM-EVs on the malignancy and progression of glioblastoma multiforme (GBM) through miRNA-mediated communication and its possible signaling pathway. Methods: The GBM and LGG data from The Cancer Genome Atlas (TCGA) were analyzed, and the immunological features of 12 clinical glioma samples, including GBM and low-grade glioma (LGG) samples, were estimated by immunohistochemistry. To evaluate the changes induced by M2-EVs compared to M1-EVs, the human GBM cell lines U87MG and A172 were cocultured with M2-EVs and M1-EVs, and their epithelial-mesenchymal transition (EMT) behaviors were investigated. The contents of M2-EVs and M1-EVs were determined by miRNA sequencing, and the differences were analyzed. Then, the EMT signatures were evaluated after a miRNA mimic was transfected into GBM cells. Six databases were used to predict the targets of the miRNA. Human GBM cell lines with interference and overexpression of the most valuable candidate miRNA targets were established. The related signaling pathways were further explored. Finally, an orthotopic homograft mouse model was employed to verify the results of the in vitro experiments. Results: The immunosuppressive GAM genes were upregulated in GBM compared with LGG, showing that the TIME of GBM is more immunosuppressive. To explore the effect of the immunosuppressive GAMs and TIME on the progression of GBM, we established immunostimulatory (M1-like) and immunosuppressive (M2-like) GAMs and compared the differences in their EVs. The migration and invasion of U87MG and A172 cells were enhanced under coculture with M2-EVs. MiR-146a-5p was deficient in M2-EVs compared with M1-EVs. Upon treatment with the miR-146a-5p mimic, the migration and invasion abilities of GBM cells were weakened, and the EMT signature was correspondingly decreased. According to the database prediction, tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 1 receptor-associated kinase 1 (IRAK1) are the targets of miR-146a-5p. TRAF6 is an E3 ubiquitin ligase, and IRAK1 is the substrate. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation assays confirmed interactions between TRAF6 and IRAK1. Interference with both TRAF6 and IRAK1 expression weakened but overexpression of both TRAF6 and IRAK1 promoted the EMT behaviors of GBM cells. The TRAF6-Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

Project description:The risk of severe disease caused by co-infection with SARS-CoV-2 and influenza virus (IAV) raises an annual concern for global public health. Extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) possess anti-inflammatory properties that can attenuate the inflammatory cytokine levels induced by viral infection. However, the effects of MSC-EV treatment on SARS-CoV-2 and IAV co-infection have not been elucidated. In the present study, we co-induced lung epithelial cells (EpiC) with SARS-CoV-2 Spike protein (S) and H1N1 influenza viral HA protein (HA) and found robust upregulation of inflammatory cytokines in comparison to those induced by either S or HA protein. Consequently, treatment of lung endothelial cells (EC) with conditioned medium from EpiC co-induced by both S and HA proteins resulted in increased apoptosis and impaired angiogenic ability, suggesting the effects of co-induction on epithelial-endothelial crosstalk. In addition, lung EpiC co-induced by both S and HA proteins showed paracrine effects on the recruitment of immune cells, including monocytes, macrophages and neutrophils. Of Note, EV derived from Wharton Jelly's MSC (WJ-EV) transferred miR-146a to recipient lung EpiC, which impaired TRAF6 and IRAK1, resulting in the downregulation of NF-κB pathway and secretion of inflammatory cytokines, rescuing the epithelial-endothelial crosstalk, and reducing the elevation of immune cell recruitment. Moreover, the anti-inflammatory properties of WJ-EV are affected by type 2 Diabetes Mellitus. WJ-EV derived from donors with type 2 Diabetes Mellitus contained less miR-146a and showed impaired ability to downregulate the NF-κB pathway and inflammatory cytokines in recipient cells. Taken together, our findings demonstrate the role of miR-146a in targeting the NF-κB pathway in the anti-inflammatory abilities of WJ-EV, which is a promising strategy to rescue the epithelial-endothelial crosstalk altered by co-infection with SARS-CoV-2 and IAV.

Project description:Rationale: Emerging evidence demonstrates that insufficient migration and invasion of trophoblasts play critical roles in the pathogenesis of recurrent spontaneous abortion (RSA). Cell-to-cell communication at the maternal-fetal interface is essential to maintain the invasion and migration of trophoblasts. M1 macrophages, important immune cellular components at the maternal-fetal interface, have been reported to be elevated in decidua tissues from patients with RSA. Recent studies indicate that M1 macrophages modulate trophoblast biological behaviors; however, the underlying mechanisms remain poorly understood. Methods: Extracellular vesicles (EVs) were isolated from the supernatant of M1 macrophages inducted from THP-1 cells (M1-EVs) by ultracentrifugation, identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting, and their miRNA profile was characterized by miRNA sequencing. Scratch wound healing and transwell assays were used to investigate the effect of M1-EVs on trophoblast migration and invasion. RT-PCR, western blotting, and luciferase reporter assays were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of M1-EVs on embryo absorption in mice. Results: M1 macrophages suppressed trophoblast EMT to reduce their migration and invasion abilities in vitro by secreting EVs. Through miRNA sequencing, miR-146a-5p and miR-146b-5p were identified as the most upregulated miRNAs in trophoblasts treated with M1-EVs. Further functional experiments showed that M1-EVs inhibited trophoblast migration and invasion by transferring miR-146a-5p and miR-146b-5p. Mechanistically, EV miR-146a-5p and miR-146b-5p inhibited EMT of trophoblasts by directly suppressing TNF receptor-associated factor 6 (TRAF6) expression at the post-transcriptional level. Furthermore, M1-EVs aggravated embryo absorption in mice. Clinically, expression of miR-146a-5p, miR-146b-5p, and TRAF6 were aberrant in placental villous tissues from patients with RSA, and negative correlations were found between miR-146a-5p/miR-146b-5p and TRAF6 expression levels. Conclusions: Our findings indicate that miR-146a-5p and miR-146b-5p derived from EVs play important roles in intercellular communication between M1 macrophages and trophoblasts, illuminating a novel mechanism in M1 macrophage regulation of trophoblasts and their role in RSA.

Dataset Information

MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway.

Publications

MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway.

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