Sonntag, Juli 31, 2022
StartHealth SciencemicroRNA-27a-3p delivered by extracellular vesicles from glioblastoma cells induces M2 macrophage polarization...

microRNA-27a-3p delivered by extracellular vesicles from glioblastoma cells induces M2 macrophage polarization through the EZH1/KDM3A/CTGF axis


GBM-EVs induce M2 macrophage polarization below hypoxia situation

To discover whether or not GBM-EVs promote M2 macrophage polarization below hypoxia situation, GBM-EVs had been extracted from the supernatant of cultured human GBM cell line U87MG, and additional recognized by transmission electron microscope (TEM), nano-particle monitoring evaluation (NTA) and Western blot evaluation. The outcomes confirmed that GBM-EVs had been typical spherical particles expressing CD63 and TSG101 proteins as a substitute of calnexin proteins, with a mean diameter of 103 ± 6.1 nm (Fig. 1A–C).

Fig. 1: GBM-EVs promote M2 polarization.
figure 1

A TEM outcomes of GBM-EVs (scale bar = 100 nm). B NTA outcomes of the scale distribution of EVs. C Expression of marker proteins TSG101, CD63 and Calnexin in 5 μg GBM-EVs and 20 μg cell lysates decided utilizing Western blot evaluation. D Immunofluorescence pictures of EV uptake by macrophages below normoxia/hypoxia (double label; purple represents EVs; inexperienced signifies macrophage) (scale bar = 25 μm). E RT-qPCR outcomes of IL-10 and TNF-α mRNA expression in macrophages. F ELISA outcomes of the secretion ranges of IL-10 and TNF-α within the supernatant of macrophages. *p < 0.05 in contrast with the traditional cells handled with PBS. # p < 0.05 in contrast with regular cells handled with GBM-EVs. The experiment was repeated 3 occasions independently.

In the meantime, human monocyte cell line THP-1 was handled with phorbol-myristate-acetate (PMA) to induce the differentiation into macrophages. Following that, macrophages had been stained with carboxyfluorescein succinimidyl ester (CFSE) whereas GBM-EVs had been stained with deep purple staining answer. Subsequently, the stained GBM-EVs had been co-cultured with CFSE-stained macrophages below normoxia or hypoxia situations, respectively. After 24-h co-culture, it was discovered via the fluorescence microscope that macrophages internalized GBM-EVs, and extra GBM-EVs had been internalized by macrophages below hypoxia situation (Fig. 1D).

As measured by reverse transcription quantitative polymerase chain response (RT-qPCR) and Enzyme-linked immunosorbent assay (ELISA), GBM-EVs below hypoxic considerably elevated the expression (Regular + GBM-EVs vs. Regular + PBS, 1.4-fold, p = 0.04, n = 3; Hypoxia + GBM-EVs vs. Regular + GBM-EVs, 1.8-fold, p < 0.01, n = 3) and secretion (Regular + GBM-EVs vs. Regular + PBS, 1.3-fold, p = 0.02, n = 3; Hypoxia + GBM-EVs vs. Regular + GBM-EVs, 3.6-fold, p < 0.01, n = 3) of interlukin-10 (IL-10) in macrophages, however prominently diminished the expression (Regular + GBM-EVs vs. Regular + PBS, 0.5-fold, p = 0.02, n = 3; Hypoxia + GBM-EVs vs. Regular + GBM-EVs, 0.3-fold, p < 0.01, n = 3) and secretion (Regular + GBM-EVs vs. Regular + PBS, 0.8-fold, p = 0.04, n = 3; Hypoxia + GBM-EVs vs. Regular + GBM-EVs, 0.6-fold, p < 0.01, n = 3) of tumor necrosis factor-α (TNF-α) (Figs. 1E, F), confirming that GBM-EVs induced M2 macrophage polarization below hypoxic situation than normoxic situation.

hsa-miR-27a-3p is transferred from GBM cells to macrophages through EVs

Moreover, elevated hsa-miR-27a-3p expression was present in GBM-related microarray GSE65626 (p < 0.01, Fig. 2A), which was additionally confirmed by RT-qPCR dedication in scientific specimens (2.3-fold, p < 0.01, Fig. 2B). Moreover, elevated hsa-miR-27a-3p expression was noticed in macrophages uncovered to GBM-EVs relative to regular macrophages (Macrophage with GBM-EVs vs. Macrophage, 1.6-fold, p < 0.01, n = 3) whereas diminished hsa-miR-27a-3p expression was detected in macrophages uncovered to GBM-EVs handled with hsa-miR-27a-3p inhibitor (Macrophage with GBM-EVs-hsa-miR-27a-3p inhibitor vs. Macrophage with GBM-EVs-inhibitor NC, 0.3-fold, p < 0.01, n = 3) (Fig. 2C). We additional discovered from RT-qPCR that in contrast with macrophages below normoxia, hsa-miR-27a-3p confirmed greater expression in macrophages below hypoxia (Hypoxia vs. Regular, 1.7-fold, p = 0.04, n = 3, Fig. 2D).

Fig. 2: hsa-miR-27a-3p is transferred into macrophages from GBM cells through EVs.
figure 2

A Differential expression of hsa-miR-27a-3p in microarray GSE65626. The X-axis represents pattern sort, and the Y-axis represents expression worth. Grey field signifies regular samples (n = 3), whereas purple field signifies tumor samples (n = 3). B RT-qPCR outcomes of hsa-miR-27a-3p expression in scientific samples of sufferers with GBM (n = 50) and non-GBM sufferers (n = 20). C RT-qPCR outcomes of hsa-miR-27a-3p expression in macrophages uncovered to GBM-EVs relative to regular macrophages. D RT-qPCR outcomes of hsa-miR-27a-3p expression in macrophages cultured below normoxia/hypoxia situations following co-culture with GBM-EVs. *p < 0.05 in contrast with regular tissues, regular cells or macrophages handled with mimic-NC. #p < 0.05 in contrast with macrophages handled with GBM-EVs-inhibitor NC. The experiment was repeated 3 occasions independently.

Along with the outcomes proven in Fig. 1D, we concluded that hsa-miR-27a-3p was delivered to macrophages by GBM-EVs.

hsa-miR-27a-3p induces M2 macrophage polarization to advertise GBM cell proliferation and motility

Subsequently, miR-27a-3p mimic/miR-27a-3p inhibitor was delivered into macrophages induced by PMA from THP1 cells to reveal the motion of hsa-miR-27a-3p in macrophage polarization, adopted by culturing below normoxia/hypoxia. RT-qPCR dedication confirmed elevated IL-10 however decreased TNF-α after therapy of hsa-miR-27a-3p mimic below hypoxia/normoxia, whereas hsa-miR-27a-3p inhibitor induced the other impact (p < 0.05, n = 3, Fig. S1A–1C). As well as, dedication of expression of macrophage polarization marker inducible NOS (iNOS) and arginase-1 (Arg-1) revealed that iNOS was downregulated and Arg-1 was upregulated in macrophages transfected with hsa-miR-27a-3p mimic below hypoxia/normoxia, whereas the other results had been noticed in macrophages transfected with hsa-miR-27a-3p inhibitor (p < 0.05, n = 3, Fig. S1D, 1E). The findings concluded that hsa-miR-27a-3p induced M2 macrophage polarization below hypoxia/normoxia.

Subsequent, the proliferation capability of GBM cells co-cultured with macrophages was strengthened after hsa-miR-27a-3p mimic therapy (1.4-fold, p < 0.01, n = 3), however weakened in GBM cells co-cultured with hsa-miR-27a-3p inhibitor-treated macrophages (1.7-fold, p < 0.01, n = 3) (Fig. S1F). Additional CFSE staining assay revealed elevated proliferating cell ratio of GBM cells co-cultured with hsa-miR-27a-3p mimic-treated macrophages (1.7-fold, p < 0.01, n = 3), whereas proliferating cell ratio of GBM cells was discovered to be diminished after co-culture with hsa-miR-27a-3p inhibitor-treated macrophages (5.8-fold, p < 0.01, n = 3) (Fig. S2A).

As well as, the outcomes of Transwell assay confirmed greater migration and invasion of GBM cells co-cultured with hsa-miR-27a-3p mimic-treated macrophages, whereas cell migration and invasion was curtailed in GBM cells co-cultured with hsa-miR-27a-3p inhibitor-treated macrophages (p < 0.01, n = 3) (Fig. S1G).

Thus, hsa-miR-27a-3p promoted the polarization of M2 macrophages, thereby facilitating the GBM cell proliferation and motility.

EZH1 is a goal gene of hsa-miR-27a-3p

We additional predicted the downstream goal genes of hsa-miR-27a-3p via starBase and mirDIP databases, revealing 4365 and 6224 genes respectively. In the meantime, differential evaluation was carried out on the microarrays GSE12657, GSE104291 and GSE50161 utilizing “limma” bundle of R language (|logFoldChange| > 1, p < 0.05), revealing 864, 3628 and 1797 differentially expressed genes respectively (Fig. 3A–C). After intersection, 141 candidate genes had been obtained (Fig. 3D, Desk S1) adopted by heatmap evaluation of their expression in three microarrays (Fig. S3). Subsequently, Gene Ontology (GO) purposeful evaluation of intersected genes revealed that these genes had been primarily enriched within the pathways of “modulation of chemical synaptic transmission”, “neuron to neuron synapase” and “protein serine/threonine kinase exercise” (Fig. 3E, Desk S2). Among the many GO pathway enrichment outcomes, pathways associated to mind growth commanded our consideration, together with telencephalon growth, forebrain growth and pallium growth. Genes concerned within the pathways consisted of SLC1A2, EZH1, CDK5R1, ERBB4, PLCB1, SLIT2, CNTNAP2, AGTPBP1, FGF13, and RAPGEF2. The candidate genes had been checked for differential expression in TCGA_LGG and GTEx (Fig. 3F), outcomes of which confirmed that EZH1, SLIT2, CNTNAP2, AGTPBP1 and FGF13 had been considerably downregulated.

Fig. 3: hsa-miR-27a-3p targets EZH1.
figure 3

A Volcano plots displaying the differentially expressed genes retrieved from microarray dataset GSE12657. B Volcano plots displaying the differentially expressed genes retrieved from microarray dataset GSE104291. C Volcano plots displaying the differentially expressed genes retrieved from microarray dataset GSE50161. The X-axis represents -log10 (p worth), the Y-axis represents logFoldChange, purple dots signify upregulated genes, and inexperienced dots point out downregulated genes. D Venn diagram displaying the intersection of goal genes of hsa-miR-27a-3p predicted by bioinformatics evaluation and differentially expressed genes retrieved from microarray datasets GSE12657, GSE104291 and GSE50161. E GO purposeful enrichment evaluation on the intersected genes. X-axis represents gene ratio. Y-axis represents GO entries. The best histogram is shade gradation. F Expression degree of candidate genes in TCGA_LGG and GTEx. G The expression of EZH1 in microarray GSE50161. H The expression of EZH1 in scientific samples of sufferers with GBM (n = 50) and non-GBM sufferers (n = 20), *p < 0.05 in contrast with regular tissues. I The binding websites between hsa-miR-27a-3p and EZH1 3’UTR in human and mice predicted by starBase databse. J The concentrating on relationship between miR-27a and EZH1 verified by twin luciferase reporter gene assay, *p < 0.05 in contrast with EZH1 3’UTR-WT + mimic-NC group. The experiment was repeated 3 occasions independently.

EZH1 expression was then analyzed within the three GBM-related microarray dataset GSE50161, exhibiting that EZH1 expression was diminished in GBM (Fig. 3G). RT-qPCR knowledge was confirmatory, exhibiting a lower in EZH1 expression in sufferers with GBM (3.3-fold, p < 0.01) (Fig. 3H).

Based on the prediction of starBase, there existed binding websites between hsa-miR-27a-3p/mmu-miR-27a-3p and EZH1 (Fig. 3I), which was verified utilizing luciferase assay that the luciferase depth of the cells co-transfected with hsa-miR-27a-3p mimic and EZH1 3’untranslated area (3’UTR)-wild sort (WT) decreased considerably (p < 0.01, n = 3) but that of the cells co-transfected with hsa-miR-27a-3p mimic and EZH1 3’UTR-mutant sort (MUT) didn’t differ considerably (p > 0.05, n = 3) (Fig. 3J).

Taken collectively, these findings indicated that EZH1 was poorly expressed in GBM and hsa-miR-27a-3p focused EZH1.

GBM-EV-hsa-miR-27a-3p downregulates EZH1 to advertise M2 macrophage polarization in contribution to GBM cell proliferation and motility

To additional validate the regulation of EZH1 by GBM-EV-hsa-miR-27a-3p on the mobile degree, U87MG cells had been transfected with hsa-miR-27a-3p mimic, adopted by the extraction of EVs. Then, EVs had been co-cultured with macrophages overexpressing EZH1 below hypoxia. We discovered an enhancement in hsa-miR-27a-3p and IL-10 expression however a discount in EZH1 and TNF-α expression within the hsa-miR-27a-3p mimic-treated macrophages. Nevertheless, the expression of hsa-miR-27a-3p didn’t considerably change within the macrophages co-transfected with hsa-miR-27a-3p mimic and EZH1 overexpression vector (oe-EZH1) and macrophages co-transfected with hsa-miR-27a-3p and oe-NC, whereas amongst hsa-miR-27a-3p mimic- and oe-EZH1-treated cells, the expression of EZH1 and TNF-α elevated and IL-10 expression decreased (p < 0.05, n = 3) (Fig. S4A–C). As well as, iNOS expression was decreased (2.3-fold, p < 0.01, n = 3) and Arg-1 expression was elevated (1.9-fold, p < 0.01, n = 3) after the therapy of hsa-miR-27a-3p mimic, whereas the overexpression of EZH1 reversed the results of hsa-miR-27a-3p mimic (Fig. S4D). The findings recommended that GBM-EV-hsa-miR-27a-3p induced M2 macrophage polarization by downregulating EZH1.

Additional CFSE staining assay confirmed that proliferating cell ratio was elevated in presence of hsa-miR-27a-3p mimic (1.7-fold, p < 0.01, n = 3) but decreased by further supply of oe-EZH1 (1.6-fold, p = 0.02, n = 3) (Fig. S2B). Then, the proliferative, migrative and invasive capabilities of GBM cells had been elevated after hsa-miR-27a-3p mimic, however had been distinctly suppressed after the addition of oe-EZH1, indicating that the overexpressed EZH1 offset the results of overexpressed hsa-miR-27a-3p on the proliferation, migration and invasion of GBM cells (p < 0.05, n = 3) (Figs. S4E, F).

The aforementioned outcomes demonstrated that EVs-hsa-miR-27a-3p induced M2 macrophage polarization by concentrating on EZH1 in contribution to GBM cell proliferation, migration and invasion.

EZH1 inhibits M2 macrophage polarization by suppressing KDM3A expression

As ChIP-Seq knowledge evaluation outcomes unraveled, EZH1 was discovered to bind to the promoter of KDM3A (Fig. 4A), suggesting that EZH1 inhibited KDM3A at a transcriptional degree. Subsequent, we carried out chromatin immunoprecipitation (ChIP) evaluation to detect whether or not EZH1 mediated expression of KDM3A via regulation on H3K27me3 enrichment on the promoter. It was discovered that overexpressed EZH1 considerably enhanced H3K27me3 enrichment on the promoter area of KDM3A (1.5-fold, p < 0.01, n = 3) (Fig. 4B). Furthermore, brief interfering RNA (siRNA) in opposition to EZH1 (si-EZH1) considerably diminished H3K27me3 enrichment on the promoter area of KDM3A gene (0.4-fold, p < 0.01, n = 3) (Fig. 4C).

Fig. 4: EZH1 suppresses M2 polarization by downregulating KDM3A.
figure 4

A ChIP-Seq knowledge evaluation outcomes of the binding websites. B Enrichment of EZH1 and H3K27me3 on the KDM3A promoter in macrophages detected by ChIP evaluation. C KDM3A mRNA expression in macrophages decided by RT-qPCR and H3K27me3 enrichment on the KDM3A promoter in macrophages detected by ChIP evaluation. D mRNA expression of EZH1 in macrophages cultured below hypoxic situations decided by RT-qPCR. E mRNA expression of KDM3A in macrophages cultured below hypoxic situations decided by RT-qPCR. F mRNA expression of IL-10 and TNF-α in macrophages cultured below hypoxic situations decided by RT-qPCR. G The expression of iNOS and Arg-1 detected by Western blot evaluation. *p < 0.05 in contrast with IgG, si-NC or si-NC + sh-NC. # p < 0.05 compared with si-EZH1 + sh-NC. The experiment was repeated 3 occasions independently.

To additional discover the regulatory relationship between EZH1 and KDM3A, EZH1 or/and KDM3A was knocked down in macrophages. Outcomes of RT-qPCR displayed that silencing EZH1 alone upregulated KDM3A and IL-10 however decreased TNF-α expression to induce M2 macrophage polarization. Whereas knockdown of KDM3A reversed the results of silenced EZH1 on M2 macrophage polarization when co-transfected with si-EZH1 (p < 0.01, n = 3) (Fig. 4D–F). Moreover, the outcomes of Western blot evaluation exhibited that silencing EZH1 downregulated the expression of iNOS (0.4-fold, p < 0.01, n = 3) and upregulated the expression of Arg-1 (2.0-fold, p < 0.01, n = 3), whereas KDM3A knockdown reversed the results of silenced EZH1 on M2 polarization (Fig. 4G).

These outcomes recommended that EZH1 inhibited M2 macrophage polarization by inhibiting KDM3A expression via H3K27me3 enrichment.

KDM3A facilitates macrophage polarization by upregulating CTGF

ChIP evaluation was then carried out utilizing antibodies to CTGF, H3K27ac and H3K4me1 after therapy of macrophages with si-KDM3A. It was discovered that KDM3A protein was extremely enriched within the enhancer area of CTGF gene (1.5-fold, p = 0.02, n = 3) and inhibited the enrichment of H3K4me1 (0.4-fold, p < 0.05, n = 3) and H3K27ac (0.4-fold, p < 0.05, n = 3) within the enhancer area (Fig. 5A, B). Additionally, knockdown of KDM3A diminished CTGF expression degree (0.2-fold, p < 0.05, n = 3) (Fig. 5B).

Fig. 5: KDM3A upregulates CTGF expression to spice up M2 polarization.
figure 5

A ChIP evaluation outcomes on enrichment of H3K4me1 and H3K27ac within the CTGF enhancer area of macrophages after addition of KDM3A. B ChIP evaluation outcomes on enrichment of H3K4me1 and H3K27ac within the CTGF enhancer area of macrophages after knockdown of KDM3A. C mRNA expression of KDM3A in macrophages cultured below hypoxic situations decided by RT-qPCR. D mRNA expression of CTGF in macrophages cultured below hypoxic situations decided by RT-qPCR. E mRNA expression of IL-10 and TNF-α in macrophages cultured below hypoxic situations decided by RT-qPCR. F Western blot evaluation outcomes of the protein expression of iNOS and Arg-1. *p < 0.05 in contrast with IgG, si-NC or oe-NC + si-NC. #p < 0.05 compared with oe-KDM3A + si-NC. The experiment was repeated 3 occasions independently.

To additional discover the regulatory relationship between KDM3A and CTGF, KDM3A was overexpressed or/and CTGF was knocked down in macrophages. Outcomes of RT-qPCR exhibited that overexpression of KDM3A upregulated CTGF (2.7-fold, p < 0.05, n = 3) and IL-10 (2.8-fold, p < 0.05, n = 3) expression however decreased TNF-α expression (0.3-fold, p < 0.05, n = 3). Nevertheless, after overexpressing KDM3A or knocking CTGF down in macrophages, it was discovered that depleted CTGF inhibited M2 macrophage polarization which was induced by overexpressed KDM3A (Fig. 5C–E). Furthermore, overexpression of KDM3A downregulated iNOS protein expression (0.4-fold, p < 0.05, n = 3) and upregulated Arg-1 protein expression (2.1-fold, p < 0.05, n = 3), whereas knockdown of CTGF reversed the M2 macrophage polarization induced by overexpression of KDM3A (Fig. 5F).

These outcomes recommended that KDM3A promoted M2 macrophage polarization by binding to CTGF enhancer areas and selling CTGF gene expression via inhibition on enrichment of H3K4me1 and H3K27ac.

GBM-EVs-released hsa-miR-27a-3p induces M2 macrophage polarization to advertise GBM cell proliferation, migration and invasion by EZH1/KDM3A/CTGF in vitro

To find out whether or not EZH1 can regulate CTGF expression via KDM3A, EZH1 or CTGF was knocked down in macrophages, adopted by the dedication of EZH1, KDM3A and CTGF expression. We discovered decreased EZH1 and TNF-α expression however upregulated KDM3A, CTGF and IL-10 after EZH1 was silenced. Nevertheless, the expression of EZH1 and KDM3A didn’t change considerably within the si-EZH1 + sh-CTGF group in contrast with the si-EZH1 + sh-NC group, whereas the expression of CTGF (0.3-fold, p < 0.05, n = 3) and IL-10 (0.5-fold, p < 0.05, n = 3) decreased after EZH1 and CTGF had been each silenced, together with elevated TNF-α expression (2.9-fold, p < 0.05, n = 3), indicating that knockdown of CTGF reversed the M2 macrophage polarization induced by overexpressed KDM3A and silenced EZH1 (Fig. 6A–D).

Fig. 6: GBM-EV-derived hsa-miR-27a-3p targets EZH1 to mediate M2 polarization via KDM3A/CTGF in vitro.
figure 6

A mRNA expression of EZH1 in macrophages after EZH1 and CTGF knockdown decided utilizing RT-qPCR. B mRNA expression of KDM3A in macrophages after EZH1 and CTGF knockdown decided utilizing RT-qPCR. C mRNA expression of CTGF in macrophages after EZH1 and CTGF knockdown decided utilizing RT-qPCR. D mRNA expression of IL-10 and TNF-α in macrophages after EZH1 and CTGF knockdown decided utilizing RT-qPCR. E mRNA expression of EZH1 in macrophages overexpressing CTGF evaluated utilizing RT-qPCR. F mRNA expression of KDM3A in macrophages overexpressing CTGF evaluated utilizing RT-qPCR. G mRNA expression of CTGF in macrophages overexpressing CTGF evaluated utilizing RT-qPCR. H Expression of hsa-miR-27a-3p after macrophages had been transfected with hsa-miR-27a-3p mimic/inhibitor decided utilizing RT-qPCR. I Protein expression of EZH1, KDM3A and CTGF in macrophages measured utilizing Western blot evaluation. J mRNA expression of IL-10 and TNF-α in macrophages detected utilizing RT-qPCR. Okay Protein expression of iNOS and Arg-1 in macrophages decided utilizing Western blot evaluation. L CCK-8 assay outcomes of proliferation capability of GBM cells. M Transwell assay outcomes of the migration and invasion capability of GBM cells (scale bar = 50 μm). *p < 0.05 in contrast with si-NC + sh-NC, oe-NC or oe-CTGF + EVs-mimic-NC. #p < 0.05 compared with si-EZH1 + sh-NC or oe-CTGF + EVs-inhibitor-NC. The experiment was repeated 3 occasions independently.

In an effort to additional decide that GBM-EVs-derived hsa-miR-27a-3p functioned as an upstream regulator of EZH1/KDM3A/CTGF in M2 macrophage polarization, CTGF was overexpressed in macrophages, adopted by the dedication of hsa-miR-27a-3p/EZH1/KDM3A/CTGF expression in every group. We noticed that oe-CTGF exerted no impact on the expression of hsa-miR-27a-3p, EZH1, and KDM3A. Nevertheless, CTGF and IL-10 expression was elevated and TNF-α expression was decreased after CTGF was overexpressed (Fig. 6E–G). EVs had been extracted from hsa-miR-27a-3p mimic/inhibitor handled GBM cells, which had been co-cultured with oe-CTGF-treated macrophages. The outcomes confirmed that in EVs extracted from hsa-miR-27a-3p mimic-treated GBM cells, upregulated CTGF prominently stimulated M2 macrophage polarization, through which the expression of hsa-miR-27a-3p (4.5-fold, p < 0.05, n = 3), KDM3A (2.2-fold, p < 0.05, n = 3), IL-10 (2.6-fold, p < 0.05, n = 3) and CTGF (2.2-fold, p < 0.05, n = 3) elevated, however EZH1 (0.3-fold, p < 0.05, n = 3) and TNF-α (0.4-fold, p < 0.05, n = 3) expression diminished. In distinction, within the EVs extracted from hsa-miR-27a-3p inhibitor-treated cells, upregulated CTGF by oe-CTGF dampened M2 macrophage polarization accompanied with downregulated hsa-miR-27a-3p (0.3-fold, p < 0.05, n = 3), KDM3A (0.2-fold, p < 0.05, n = 3), CTGF (0.2-fold, p < 0.05, n = 3) and IL-10 (0.4-fold, p < 0.05, n = 3) expression however upregulated EZH1 (2.3-fold, p < 0.05, n = 3) and TNF-α expression (2.2-fold, p < 0.05, n = 3) (Fig. 6H–J). Moreover, expression of iNOS decreased (0.4-fold, p < 0.05, n = 3) and Arg-1 elevated (2.2-fold, p < 0.05, n = 3) within the GBM-EVs containing hsa-miR-27a-3p mimic and macrophages overexpressing CTGF. Nevertheless, the outcomes had been reversed within the GBM-EVs containing hsa-miR-27a-3p inhibitor and macrophages overexpressing CTGF (iNOS: 2.2-fold, p < 0.05, n = 3; Arg-1: 0.4-fold, p < 0.05, n = 3) (Fig. 6K).

The function of conditioned macrophages within the GBM cell organic processes was additional explored in vitro. We noticed that in GBM-EVs containing hsa-miR-27a-3p mimic and macrophages overexpressing CTGF, strengthened proliferation (1.5-fold, p < 0.05, n = 3), invasion (1.5-fold, p < 0.05, n = 3) and migration (1.6-fold, p < 0.05, n = 3) skills was noticed, which was abolished in GBM-EVs containing hsa-miR-27a-3p inhibitor and macrophages overexpressing CTGF (proliferation: 2.0-fold, p < 0.05, n = 3; migration: 2.0-fold, p < 0.05, n = 3; invasion: 2.2-fold, p < 0.05, n = 3) (Fig. 6L, M).

Thus, GBM-EVs carrying hsa-miR-27a-3p boosted M2 macrophage polarization and finally facilitated the proliferative, migrative and invasive capabilities of GBM cells through the EZH1/KDM3A/CTGF axis.

GBM-EV-derived hsa-miR-27a-3p induces M2 macrophage polarization to irritate GBM development in vivo

To validate the above-mentioned leads to vivo, nude mice had been injected with EVs from GBM cells overexpressing hsa-miR-27a-3p or/and macrophages transfected with lentivirus-mediated CTGF to develop xenograft tumors. The outcomes of RT-qPCR confirmed greater transfection effectivity of hsa-miR-27a-3p and CTGF (p < 0.01, n = 6) (Fig. 7A).

Fig. 7: GBM-EV-derived hsa-miR-27a-3p aggravates GBM in vivo.
figure 7

Nude mice had been injected with EVs from GBM cells overexpressing hsa-miR-27a-3p or/and macrophages transfected with lentivirus-mediated CTGF. A hsa-miR-27a-3p expression and CTGF mRNA expression in every group of mice (n = 6) decided utilizing RT-qPCR. B H&E staining outcomes of GBM cell invasion to the mouse mind tissues (n = 6) (scale bar = 50 μm). C Statistical knowledge of survival time of mice in every group (n = 6). D Western blot evaluation outcomes of the protein expression of EZH1, KDM3A and CTGF in mouse mind tissues of every group (n = 6). E ELISA outcomes of secretion ranges of IL-10 and TNF-α in peripheral serum of mice (n = 6). F ELISA outcomes of content material ranges of IL-10 and TNF-α in lysate of mouse tumor xenografts (n = 6). *p < 0.05 in contrast with sham-operated mice. #p < 0.05 in contrast with mice harboring EVs containing mimic-NC. & p < 0.05 in contrast with mice harboring EVs containing hsa-miR-27a-3p mimic + sh-NC.

Hematoxylin-Eosin (H&E) staining exhibited that the boundary of mouse mind tissues was blurred after therapy of GBM-EVs harboring hsa-miR-27a-3p mimic, accompanied with elevated invasive capability of GBM cells. Whereas the invasive capability of tumors to mind tissues was weaker in mice harboring hsa-miR-27a-3p mimic-treated GBM-EVs after CTGF expression was silenced (Fig. 7B). As well as, the survival time of mice was shortened after GBM-EVs overexpressing hsa-miR-27a-3p had been injected into mice, whereas the survival time of mice was extended after interfering with CTGF (Fig. 7C).

Moreover, GBM-EVs carrying hsa-miR-27a-3p mimic led to downregulated EZH1 (0.8-fold, p = 0.03, n = 6) and upregulated KDM3A (1.9-fold, p < 0.01, n = 6) and CTGF (1.7-fold, p < 0.01, n = 6) in mouse mind tissues. Nevertheless, after silencing of CTGF, the expression of EZH1 and KDM3A didn’t change considerably, whereas the expression of CTGF was downregulated (0.5-fold, p < 0.01, n = 6) (Fig. 7D). Furthermore, GBM-EVs carrying overexpressed hsa-miR-27a-3p resulted within the improve of IL-10 (2.3-fold, p < 0.01, n = 6) and the lower of TNF-α (0.5-fold, p < 0.01, n = 6) in peripheral serum of mice, indicating that M2 polarization was induced in mice. Nevertheless, the lower of IL-10 (0.7-fold, p < 0.01, n = 6) and the rise of TNF-α (1.4-fold, p < 0.01, n = 6) had been noticed after silencing of CTGF, suggesting that lack of CTGF prevented M2 macrophages polarization to a point (Fig. 7E). As well as, ELISA in tumor xenograft revealed upregulation of IL-10 (1.8-fold, p < 0.01, n = 6) and downregulation of TNF-α (0.7-fold, p < 0.01, n = 6) in presence of hsa-miR-27a-3p mimic whereas further therapy of brief hairpin RNA (shRNA) in opposition to CTGF (sh-CTGF) led to a discount in IL-10 (0.7-fold, p < 0.01, n = 6) and a rise in (1.6-fold, p < 0.01, n = 6) (p < 0.01, n = 6) (Fig. 7F).

These outcomes recommended that GBM-EVs-derived hsa-miR-27a-3p promoted the event of GBM in vivo by induces M2 macrophage polarization.

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