miR-365 secreted from M2 Macrophage-derived extracellular vesicles promotes pancreatic ductal adenocarcinoma progression through the BTG2/FAK/AKT axis

Clinical and experimental evidence indicates that tumour-associated macrophages support cancer progression. Moreover, macrophage-derived extracellular vesicles (EVs) are involved in pathogenesis of multiple cancers, yet the functions of molecular determinants in which have not been fully understood. Herein, we aim to understand whether macrophage modulates pancreatic ductal adenocarcinoma (PDAC) progression in an EV-dependent manner and the underlying mechanisms. microRNA (miR)-365 was experimentally determined to be enriched in the EVs from M2 macrophages (M2-EVs), which could be transferred into PDAC cells. Using a co-culture system, M2-EVs could enhance the proliferating, migrating and invading potentials of PDAC cells, while inhibition of miR-365 in M2-EVs could repress these malignant functions. B-cell translocation gene 2 (BTG2) was identified to be a direct target of miR-365, while the focal adhesion kinase (F/ATP)-dependent tyrosine kinase (AKT) pathway was activated by miR-365. We further demonstrated that overexpression of BTG2 could delay the progression of PDAC in vitro, whereas by impairing BTG2-mediated anti-tumour effect, M2-EV-miR-365 promoted PDAC progression. For validation, a nude mouse model of tumorigenesis was established, in which we found that targeting M2-EV-miR-365 contributed to suppression of tumour growth. Collectively, M2-EVs carry miR-365 to suppress BTG2 expression, which activated FAK/AKT pathway, thus promoting PDAC development.     

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.     

CircRNA circPDSS1 promotes the gastric cancer progression by sponging miR-186-5p and modulating NEK2

The aim of this study is to investigate the regulatory mechanism of circPDSS1/miR-186-5p/NEK2 axis on the viability and proliferation in gastric cancer (GC) cell line. Differentially expressed circRNAs, miRNAs, and mRNAs in GC tissues and paracarcinoma tissues were analyzed using gene chips GSE83521, GSE89143, and GSE93415. Then, the expression of circPDSS1, miR-186-5p, and NEK2 was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was adopted to explore the association between the circPDSS1 expression and the prognosis of GC. The effect of circPDSS1 on GC cell cycle and apoptosis was verified with the flow cytometry. Targeting relationships among circPDSS1, miR-186-5p, and NEK2 were predicted via bioinformatics analysis and demonstrated by the dual-luciferase reporter assay. Our results showed that circPDSS1 and NEK2 were high-expressed whereas miR-186-5p was low-expressed in GC tissues and cells. CircPDSS1 promoted GC cell cycle and inhibited apoptosis by sponging miR-186-5p, while miR-186-5p inhibited cell cycle and promoted apoptosis by targeting NEK2. Thus, circPDSS1 acts as a tumor promoter by regulating miR-186-5p and NEK2, which could be a potential biomarker and therapeutic target for the management of GC.     

Functional delivery of lncRNA TUG1 by endothelial progenitor cells derived extracellular vesicles confers anti-inflammatory macrophage polarization in sepsis via impairing miR-9-5p-targeted SIRT1 inhibition

The delivery of biomolecules by extracellular vesicles (EVs) derived from endothelial progenitor cells (EPCs) has been proven to ameliorate sepsis, yet the therapeutic mechanism remains to be elucidated. Taurine upregulated gene 1 (TUG1) is a long noncoding RNA (lncRNA) that is downregulated in sepsis. The current study was designed to explore the role of EPCs derived EVs transmitting TUG1 in macrophage polarization and macrophage-mediated inflammation in a cecal ligation and puncture (CLP)-induced sepsis mouse model. TUG1 was underexpressed in CLP-induced sepsis, and its reexpression induced anti-inflammatory macrophage polarization and suppressed macrophage-medicated inflammatory injury to the pulmonary vascular endothelium. EPCs derived EVs transmitted TUG1 to promote M2 macrophage polarization. Luciferase, RIP, and RNA pull-down assays showed that TUG1 could competitively bind to microRNA-9-5p (miR-9-5p) to upregulate the expression of sirtuin 1 (SIRT1). Furthermore, EPCs derived EVs transmitted TUG1 to promote M2 macrophage polarization through the impairment of miR-9-5p-dependent SIRT1 inhibition. Finally, EPCs derived EVs carrying TUG1 were verified to ameliorate sepsis-induced organ damage in the murine model. In summary, EPCs derived EVs transmit TUG1 to attenuate sepsis via macrophage M2 polarization. This study also highlights the proinflammatory mechanism associated with miR-9-5p-mediated inhibition of SIRT1, which contributes to a more comprehensive understanding of the pathogenesis of sepsis.Subject terms: Cell biology, Diseases     

Lipotoxic hepatocytes promote nonalcoholic fatty liver disease progression by delivering microRNA-9-5p and activating macrophages

M1-polarized macrophages are involved in chronic inflammatory diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the activation of macrophages in NAFLD have not been fully elucidated. This study aimed at investigating the physiological mechanisms by which extracellular vesicles (EVs)-encapsulated microRNA-9-5p (miR-9-5p) derived from lipotoxic hepatocytes might activate macrophages in NALFD. After blood sample and cell collection, EVs were isolated and identified followed by co-culture with macrophages. Next, the palmitic acid-induced cell and high fat diet-induced mouse NALFD models were established to explore the in vitro and in vivo effects of EVs-loaded miR-9-5p on NAFLD as evidenced by inflammatory cell infiltration and inflammatory reactions in macrophages. Additionally, the targeting relationship between miR-9-5p and transglutaminase 2 (TGM2) was identified using dual-luciferase reporter gene assay. miR-9-5p was upregulated in the NAFLD-EVs, which promoted M1 polarization of THP-1 macrophages. Furthermore, miR-9-5p could target TGM2 to inhibit its expression. Downregulated miR-9-5p in NAFLD-EVs alleviated macrophage inflammation and M1 polarization as evidenced by reduced levels of macrophage inflammatory factors, positive rates of CD86⁺ CD11b⁺, and levels of macrophage surface markers in vitro. Moreover, the effect of silencing of miR-9-5p was replicated in vivo, supported by reductions in TG, TC, AST and ALT levels and attenuated pathological changes. Collectively, lipotoxic hepatocytes-derived EVs-loaded miR-9-5p downregulated the expression of TGM2 and facilitated M1 polarization of macrophages, thereby promoting the progression of NAFLD. This highlights a potential therapeutic target for treating NAFLD.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

LINC00958 facilitates cervical cancer cell proliferation and metastasis by sponging miR-625-5p to upregulate LRRC8E expression

Accepted as a malignant tumor worldwide, cervical cancer (CC) has attracted much attention for its high incidence and mortality rates. Previous studies have elucidated the critical regulatory function that long noncoding RNAs (lncRNAs) exert on the tumorigenesis and progression of diverse tumors. Although multiple investigations have depicted that LINC00958 has a great impact on the complex biological process of many cancers, knowledge concerning the regulatory role of LINC00958 in CC remains limited and needs to be further explored. In our study, LINC00958 expression was evidently overexpressed in CC tissues and cells. Besides this, LINC00958 negatively regulated miR-625-5p expression and was verified to bind with miR-625-5p in CC. Subsequently, it was testified by a series of experiments that LINC00958 promotes CC cell proliferation and metastasis by sponging miR-625-5p. Furthermore, the leucine-rich repeat containing the eight family member E (LRRC8E) could bind with miR-625-5p, and its expression was negatively modulated by miR-625-5p, whereas positively regulated by LINC00958 in CC. Final rescue assays verified the effects of LINC0095/LRRC8E interaction and miR-625-5p/LRRC8E interaction on CC cell proliferation and metastasis. Collectively, LINC00958 facilitates CC cell proliferation and metastasis via the miR-625-5p/LRRC8E axis.     

Extracellular vesicles extracted from bone marrow mesenchymal stem cells carrying MicroRNA-342-3p inhibit the INHBA/IL13Rα2 axis to suppress the growth and metastasis of breast cancer

Increasing focus has come to the role of extracellular vesicles (EVs) in various cancers. Hence, we designed this study to explore the mechanism whereby microRNA-342-3p (miR-342-3p)-containing EVs derived from BMSCs might affect breast cancer. MCF-7 breast cancer cell line was co-incubated with the EVs isolated from rat BMSCs, followed by alteration of miR-342-3p and INHBA expression. Microarray-based analyses predicted a possible regulatory mechanism involving miR-342-3p, INHBA, and IL13Rα2 in breast cancer, which was verified by luciferase reporter, RNA pull-down, and RIP assays. Besides, in order to evaluate the effects of miR-342-3p on the biological features of breast cancer cells in vitro and in vivo, we employed the scratch assay, Transwell assay, CCK-8 assay, and nude mouse tumorigenicity assay. miR-342-3p carried by BMSC-EVs was transferred into breast cancer cells through co-culture, which inhibited the proliferation and metastasis of breast cancer cells in vitro. miR-342-3p downregulated the expression of INHBA, which further repressed the expression of IL13Rα2. Finally, the in vivo experimental results revealed the inhibitory role of miR-342-3p in tumor growth and metastasis in nude mice. To sum up, BMSC-EVs carrying miR-342-3p could prevent breast cancer growth and metastasis by downregulating the INHBA/IL13Rα2 axis, highlighting a potential target for anti-cancer treatment for breast cancer.     

Human bone marrow mesenchymal stem cell-derived extracellular vesicles impede the progression of cervical cancer via the miR-144-3p/CEP55 pathway

Cervical cancer is the most common gynaecological malignancy, with a high incidence rate and mortality rate in middle-aged women. Human bone marrow mesenchymal stem cells (hBMSCs) have been implicated in the initiation and subsequent development of cancer, along with the involvement of extracellular vesicles (EVs) mediating intracellular communication by delivering microRNAs (miRNAs or miRs). This study is aimed at investigating the physiological mechanisms by which EVs-encapsulated miR-144-3p derived from hBMSCs might mediate the progression of cervical cancer. The expression profiles of centrosomal protein, 55 Kd (CEP55) and miR-144-3p in cervical cancer cell lines and tissues, were quantified by RT-qPCR and Western blot analysis. The binding affinity between miR-144-3p and CEP55 was identified using in silico analysis and luciferase activity determination. Cervical cancer cells were co-cultured with EVs derived from hBMSCs that were treated with either miR-144-3p mimic or miR-144-3p inhibitor. Cervical cancer cell proliferation, invasion, migration and apoptosis were detected in vitro. The effects of hBMSCs-miR-144-3p on tumour growth were also investigated in vivo. miR-144-3p was down-regulated, whereas CEP55 was up-regulated in cervical cancer cell lines and tissues. CEP55 was targeted by miR-144-3p, which suppressed cervical cancer cell proliferation, invasion and migration and promoted apoptosis via CEP55. Furthermore, similar results were obtained by hBMSCs-derived EVs carrying miR-144-3p. In vivo assays confirmed the tumour-suppressive effects of miR-144-3p in hBMSCs-derived EVs on cervical cancer. Collectively, hBMSCs-derived EVs-loaded miR-144-3p impedes the development and progression of cervical cancer through target inhibition of CEP55, therefore providing us with a potential therapeutic target for treating cervical cancer.     

Tumor-derived extracellular vesicles shuttle c-Myc to promote gastric cancer growth and metastasis via the KCNQ1OT1/miR-556-3p/CLIC1 axis

Gastric cancer (GC) is a heterogeneous disease with poor prognosis. Tumor-derived extracellular vesicles (EVs) assume a role in intercellular communication by carrying various molecules, including proteins, RNA, and DNAs, which has been identified to exhibit oncogenic effect in GC. Therefore, this research aimed to figure out whether tumor-derived EVs transmit c-Myc to orchestrate the growth and metastasis of GC. KCNQ1OT1, microRNA (miR)-556-3p and CLIC1 expression of GC tissues was detected through RT-qPCR. EVs were isolated from GC cells, followed by RT-qPCR and Western blot analysis of c-Myc expression in EVs and GC cells. Next, GC cells were incubated with EVs or transfected with a series of mimic, inhibitor, or siRNAs to assess their effects on cell viability, migrative, invasive, and apoptotic potential. Relationship among c-Myc, KCNQ1OT1, miR-556-3p, and CLIC1 was evaluated by dual-luciferase reporter assay. PI3K/AKT pathway-related proteins were assessed through Western blot analysis. KCNQ1OT1 and CLIC1 were highly expressed but miR-556-3p in GC tissues. c-Myc was high-expressed in tumor-derived EVs and GC cells. Mechanistically, c-Myc could induce KCNQ1OT1 expression, and KCNQ1OT1 bound to miR-556-3p that negatively targeted CLIC1 to inactivate PI3K/AKT pathway. Tumor-derived EVs, EVs-c-Myc, KCNQ1OT1 or CLIC1 overexpression, or miR-556-3p inhibition promoted GC cell proliferative, invasive, and migrative capacities but repressed their apoptosis through activating PI3K/AKT pathway. Collectively, tumor-derived EVs carrying c-Myc activated KCNQ1OT1 to downregulate miR-556-3p, thus elevating CLIC1 expression to activate the PI3K/AKT pathway, which facilitated the growth and metastasis of GC.Subject terms: Cancer, Biotechnology     

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. RESULTS: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cells types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p). CONCLUSION: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.     

Breast cancer stem cell-derived extracellular vesicles transfer ARRDC1-AS1 to promote breast carcinogenesis via a miR-4731-5p/AKT1 axis-dependent mechanism

ObjectivesDeregulation of long non-coding RNAs (lncRNAs) has been frequently reported in breast cancer (BC). This goes to show the importance of understanding its significant contribution towards breast carcinogenesis. In the present study, we clarified a carcinogenic mechanism based on the ARRDC1-AS1 delivered by breast cancer stem cells-derived extracellular vesicles (BCSCs-EVs) in BC.MethodsThe isolated and well characterized BCSCs-EVs were co-cultured with BC cells. The expression of ARRDC1-AS1, miR-4731-5p, and AKT1 was determined in BC cell lines. BC cells were assayed for their viability, invasion, migration and apoptosis in vitro by CCK-8, Transwell and flow cytometry, as well as tumor growth in vivo after loss- and gain-of function assays. Dual-luciferase reporter gene, RIP and RNA pull-down assays were performed to determine the interactions among ARRDC1-AS1, miR-4731-5p, and AKT1.ResultsElevation of ARRDC1-AS1 and AKT1 as well as miR-4731-5p downregulation were observed in BC cells. ARRDC1-AS1 was enriched in BCSCs-EVs. Furthermore, EVs containing ARRDC1-AS1 enhanced the BC cell viability, invasion and migration and glutamate concentration. Mechanistically, ARRDC1-AS1 elevated the expression of AKT1 by competitively binding to miR-4731-5p. ARRDC1-AS1-containing EVs were also found to enhance tumor growth in vivo.ConclusionCollectively, BCSCs-EVs-mediated delivery of ARRDC1-AS1 may promote the malignant phenotypes of BC cells via the miR-4731-5p/AKT1 axis.     

miR-21-5p in extracellular vesicles obtained from adipose tissue-derived stromal cells facilitates tubular epithelial cell repair in acute kidney injury

Background aimsAcute kidney injury (AKI) is often associated with poor patient outcomes. Extracellular vesicles (EVs) have a marked therapeutic effect on renal recovery. This study sought to explore the functional mechanism of EVs from adipose tissue-derived stromal cells (ADSCs) in tubular epithelial cell (TEC) repair in AKI.MethodsADSCs were cultured and EVs were isolated and identified. In vivo and in vitro AKI models were established using lipopolysaccharide (LPS).ResultsEVs increased human kidney 2 (HK-2) cell viability; decreased terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and levels of kidney injury molecule 1, cleaved caspase-1, apoptosis-associated speck-like protein containing a CARD, gasdermin D-N, IL-18 and IL-1β; and elevated pro-caspase-1. EVs carried miR-21-5p into LPS-induced HK-2 cells. Silencing miR-21-5p partly eliminated the ability of EVs to suppress HK-2 cell pyroptosis and inflammation. miR-21-5p targeted toll-like receptor 4 (TLR4) and inhibited TEC pyroptosis and inflammation after AKI by inhibiting TLR4. TLR4 overexpression blocked the inhibitory effects of EVs on TEC pyroptosis and inflammation. EVs suppressed the nuclear factor-κB/NOD-like receptor family pyrin domain-containing 3 (NF-κB/NLRP3) pathway via miR-21-5p/TLR4. Finally, AKI mouse models were established and in vivo assays verified that ADSC-EVs reduced TEC pyroptosis and inflammatory response and potentiated cell repair by mediating miR-21-5p in AKI mice.ConclusionsADSC-EVs inhibited inflammation and TEC pyroptosis and promoted TEC repair in AKI by mediating miR-21-5p to target TLR4 and inhibiting the NF-κB/NLRP3 pathway.     

Extracellular vesicles-derived miR-150-5p secreted by adipose-derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis

Hepatic fibrosis (HF) is involved in aggravated wound-healing response as chronic liver injury. Extracellular vesicles (EVs) carrying microRNA (miR) have been reported as therapeutic targets for liver diseases. In this study, we set out to explore whether adipose-derived mesenchymal stem cells (ADMSCs)-derived EVs containing miR-150-5p affect the progression of HF. Carbon tetrachloride (CCl₄) was firstly used to induce HF mouse models in C57BL/6J mice, and activation of hepatic stellate cells (HSCs) was achieved using transforming growth factor β (TGF-β). EVs were then isolated from ADMSCs and co-cultured with HSCs. The relationship between miR-150-5p and CXCL1 was identified using dual luciferase gene reporter assay. Following loss- and gain-function experimentation, HSC proliferation was examined by MTT assay, and levels of fibrosis-, HSC activation- and apoptosis-related genes were determined in vitro. Additionally, pathological scores, collagen volume fraction ( CVF) as well as levels of inflammation- and hepatic injury-associated genes were determined in in vivo. Down-regulated miR-150-5p and elevated CXCL1 expression levels were detected in HF tissues. ADMSCs-derived EVs transferred miR-150-5p to HSCs. CXCL1 was further verified as the downstream target gene of miR-150-5p. Moreover, ADMSCs-EVs containing miR-150-5p markedly inhibited HSC proliferation and activation in vitro. Meanwhile, in vivo experiments also concurred with the aforementioned results as demonstrated by inhibited CVF, reduced inflammatory factor levels and hepatic injury-associated indicators. Both experiments results were could be reversed by CXCL1 over-expression. Collectively, our findings indicate that ADMSCs-derived EVs containing miR-150-5p attenuate HF by inhibiting the CXCL1 expression.     

MircoRNA-29a in Astrocyte-derived Extracellular Vesicles Suppresses Brain Ischemia Reperfusion Injury via TP53INP1 and the NF-κB/NLRP3 Axis

Brain ischemia reperfusion injury (BIRI) is defined as a series of brain injury accompanied by inflammation and oxidative stress. Astrocyte-derived extracellular vesicles (EVs) are importantly participated in BIRI with involvement of microRNAs (miRs). Our study aimed to discuss the functions of miR-29a from astrocyte-derived EVs in BIRI treatment. Thus, astrocyte-derived EVs were extracted. Oxygen and glucose deprivation (OGD) cell models and BIR rat models were established. Then, cell and rat activities and pyroptosis-related protein levels in these two kinds of models were detected. Functional assays were performed to verify inflammation and oxidative stress. miR-29a expression in OGD cells and BIR rats was measured, and target relation between miR-29a and tumor protein 53-induced nuclear protein 1 (TP53INP1) was certified. Rat neural function was tested. Astrocyte-derived EVs improved miR-29a expression in N9 microglia and rat brains. Astrocyte-derived EVs inhibited OGD-induced injury and inflammation in vitro, reduced brain infarction, and improved BIR rat neural functions in vivo. miR-29a in EVs protected OGD-treated cells and targeted TP53INP1, whose overexpression suppressed the protective function of EVs on OGD-treated cells. miR-29a alleviated OGD and BIRI via downregulating TP53INP1 and the NF-κB/NLRP3 pathway. Briefly, our study demonstrated that miR-29a in astrocyte-derived EVs inhibits BIRI by downregulating TP53INP1 and the NF-κB/NLRP3 axis.