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. 2022 Nov;28(11):1733-1747.
doi: 10.1111/cns.13905. Epub 2022 Sep 2.

Hypoxic glioma-derived extracellular vesicles harboring MicroRNA-10b-5p enhance M2 polarization of macrophages to promote the development of glioma

Bingzhen Li  1 , Cheng Yang  1 , Zhanpeng Zhu  1 , Hao Chen  1 , Bin Qi  1
Affiliations

Hypoxic glioma-derived extracellular vesicles harboring MicroRNA-10b-5p enhance M2 polarization of macrophages to promote the development of glioma

Bingzhen Li et al. CNS Neurosci Ther. 2022 Nov.

Abstract

Introduction: The delivery of biomolecules by tumor cell-secreted extracellular vesicles (EVs) is linked to the development of glioma. Here, the present study was implemented to explore the functional significance of hypoxic glioma cell-derived EVs carrying microRNA-10b-5 (miR-10b-5p) on glioma with the involvement of polarization of M2 macrophages.

Methods: EVs were isolated from hypoxia-stimulated glioma cells, and their role in polarization of M2 macrophages was studied by co-culturing with macrophages. miR-10b-5p expression in glioma tissues, glioma-derived EVs, and macrophages co-cultured with EVs was characterized. Interaction among miR-10b-5p, NEDD4L, and PIK3CA was analyzed. The macrophages or glioma cells were transfected with overexpressing plasmid or shRNA to study the effects of miR-10b-5p/NEDD4L/PIK3CA on M2 macrophage polarization, and glioma cell proliferation, migration, and invasion in vitro and in vivo.

Results: Promotive role of hypoxia-stimulated glioma-derived EVs in macrophage M2 polarization was confirmed. Elevation of miR-10b-5p occurred in glioma tissues, glioma-derived EVs and macrophages co-cultured with EVs, and stimulated M2 polarization of macrophages. NEDD4L was a target gene of miR-10b-5p. Overexpression of NEDD4L could inhibit PI3K/AKT pathway through increase in ubiquitination and degradation of PIK3CA. Hypoxic glioma-derived EVs harboring upregulated miR-10b-5p triggered an M2 phenotype in macrophages as well as enhanced aggressive tumor biology of glioma cells via inhibition of PIK3CA/PI3K/AKT pathway by targeting NEDD4L.

Conclusions: In summary, miR-10b-5p delivered by hypoxic glioma-derived EVs accelerated macrophages M2 polarization to promote the progression of glioma via NEDD4L/PIK3CA/PI3K/AKT axis.

Keywords: extracellular vesicles; glioma; microRNA-10b-5; neuronal precursor cell expressed developmentally downregulated 4-like; phosphatidylinositol-4, 5-bisphosphate 3-kinase.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Hypoxic glioma cell‐derived EVs promote M2 macrophage polarization. (A) Images of EVs observed under a TEM (Scale bar = 100 nm). (B) Diameter of EVs detected by dynamic light scattering. (C) The expression of CD9, CD63, TSG101, and calnexin on the surface of EVs determined by Immunoblotting. (D) Detection of the uptake of EVs from normoxic and hypoxic glioma cells by macrophages U937 through immunofluorescence (400×). (E) The mRNA expression of iNOS, TNF‐α, Arg‐1, and IL‐10 assessed by RT‐qPCR after EV treatment. (F) Immunoblotting of protein expression of iNOS and Arg‐1 in macrophages U937 after EV treatment. (G) The expression of TNF‐α and IL‐10 in supernatant of macrophages U937 evaluated by ELISA assay. (H) The proportion of CD11b+CD163+ cells in U937 cells examined by flow cytometry. *p < 0.05 versus macrophages treated with PBS. # p < 0.05 versus macrophages treated with normal oxygen‐induced glioma cell‐derived EVs. The experiment was repeated three times
FIGURE 2
FIGURE 2
Hypoxic glioma cell‐derived EVs harboring miR‐10b‐5p facilitate M2 polarization of macrophages, thus increasing glioma development. (A) Heatmap of differential miRNAs in glioma‐related miRNA expression microarray. The x‐axis represents the sample number, the y‐axis represents the miRNA name, and the upper right histogram is color scale. (B) Intersection of differential miRNAs with miRNAs in blood EVs in EVmiRNA database. The middle part is the intersection of two sets of data. (C) The expression of miR‐10b‐5p in brain tissues of 40 cases of glioma and 15 cases of non‐glioma examined by RT‐qPCR. (D) The expression of miR‐10b‐5p in N‐EV and H‐EV determined by RT‐qPCR. (E) The expression of miR‐10b‐5p in U937 cells co‐cultured with N‐EV and H‐EV measured by RT‐qPCR. (F) The expression of miR‐10b‐5p in U937 cells treated with miR‐10b‐5p‐mimic evaluated by RT‐qPCR. (G) The mRNA expression of iNOS, TNF‐α, Arg‐1, and IL‐10 in U937 cells treated with miR‐10b‐5p‐mimic assessed by RT‐qPCR. (H) Immunoblotting of protein expression of iNOS and Arg‐1 in U937 cells treated with miR‐10b‐5p‐mimic. (I) The expression of TNF‐α and IL‐10 in U937 cell supernatant treated with miR‐10b‐5p‐mimic detected by ELISA. (J) The proportion of CD11b+CD163+ cells in U937 cells treated with miR‐10b‐5p‐mimic evaluated by flow cytometry. (K) The proliferation in glioma U87 and A172 cells co‐cultured with miR‐10b‐5p‐ mimic or mimic‐NC transfected U937 cells tested by EDU. (L) Cell migration in U87 and A172 cells co‐cultured with miR‐10b‐5p‐mimic or mimic‐NC transfected U937 cells examined by Transwell. (M) Cell invasion in U87 and A172 cells co‐cultured with miR‐10b‐5p‐mimic or mimic‐NC transfected U937 cells examined by Transwell. *p < 0.05 versus brain tissues from non‐glioma or U937 cells treated with PBS or mimic‐NC. # p < 0.05 versus U937 treated with normal oxygen‐induced glioma‐derived EVs. & p < 0.05 versus U87 or A172 co‐cultured with U937 cells without any treatment. The experiment was repeated three times
FIGURE 3
FIGURE 3
NEDD4L is negatively targeted by miR‐10b‐5p in macrophages. (A) The target site between miR‐10b‐5p and NEDD4L predicted through the online Starbase website. (B) The expression of NEDD4L in brain tissues of 40 cases of glioma and 15 cases of non‐glioma measured by RT‐qPCR. (C) The expression of NEDD4L in brain tissues of 40 cases of glioma and 15 cases of non‐glioma determined by immunohistochemistry (×200). (D) NEDD4L expression negatively correlated with miR‐10b‐5p expression revealed by Pearson correlation analysis. (E) Targeting relation between miR‐10b‐5p and NEDD4L identified by dual‐luciferase reporter gene assay. (F) The expression of miR‐10b‐5p and mRNA expression of NEDD4L in U937 cells after miR‐10b‐5p alteration assessed by RT‐qPCR. (G) Immunoblotting for determination of protein expression of miR‐10b‐5p and NEDD4L in U937 cells. *p < 0.05 versus brain tissues from patients with non‐glioma or NEDD4L 3′UTR‐WT co‐transfection with mimic‐NC. # p < 0.05 versus macrophages treated with inhibitor‐NC. The experiment was repeated three times
FIGURE 4
FIGURE 4
miR‐10b‐5p stimulates M2 polarization of macrophages to increase proliferation, migration, and invasion of glioma cells via downregulation of NEDD4L. (A) The expression of miR‐10b‐5p and NEDD4L in U937 cells after overexpression of miR‐10b‐5p and NEDD4L measured by RT‐qPCR. (B) The mRNA expression of iNOS, TNF‐α, Arg‐1, and IL‐10 in U937 cells after overexpression of miR‐10b‐5p and NEDD4L determined by RT‐qPCR. (C) The protein expression of iNOS and Arg‐1 in U937 cells after overexpression of miR‐10b‐5p and NEDD4L examined by Immunoblotting. (D) The expression of TNF‐α and IL‐10 assessed in supernatant of U937 cells after overexpression of miR‐10b‐5p and NEDD4L examined by ELISA. (E) The proportion of CD11b+CD163+ cells in U937 cells after overexpression of miR‐10b‐5p and NEDD4L evaluated by flow cytometry. (F) The proliferation in glioma U87 and A172 cells co‐cultured with transfected U937 cells assessed by EdU. (G) Cell migration in U87, and A172 cells co‐cultured with U937 cells tested by Transwell. H, Cell invasion in U87 and A172 cells co‐cultured with U937 cells tested by Transwell. *p < 0.05 versus U937 cells transfected with Vector and mimic‐NC or U87 or A172 co‐cultured with U937 cells treated without anything. # p < 0.05 versus U937 cells transfected with NEDD4L and mimic‐NC or U87 or A172 co‐cultured with U937 cells transfected with NEDD4L and mimic‐NC. The experiment was repeated three times
FIGURE 5
FIGURE 5
NEDD4L enhances ubiquitination and degradation of PIK3CA in macrophages. (A) PIK3CA‐related gene prediction. Circle center denotes PIK3CA gene, and others are predicted related genes. (B) KEGG pathway enrichment analysis of PIK3CA‐related genes. The x‐axis represents GeneRatio, the y‐axis represents the KEGG entry name, and the histogram on the right is color scale. (C) The expression of PIK3CA in brain tissues of 40 cases of glioma and 15 cases of non‐glioma examined by RT‐qPCR. (D) The expression of PIK3CA in brain tissues of 40 cases of glioma and 15 cases of non‐glioma assessed by immunohistochemistry (×200). (E) NEDD4L expression negatively correlated with PIK3CA expression identified by Pearson correlation analysis. (F) The mRNA expression of NEDD4L and PIK3CA in U937 cells evaluated by RT‐qPCR. (G) Immunoblotting for measurement of protein expression of NEDD4L and PIK3CA in U937 cells. (H) Interaction between NEDD4L and PIK3CA verified by Co‐IP assay. (I) The protein expression of NEDD4L and PIK3CA in U937 cells with 20–40 μg/ml CHX treatment determined by Immunoblotting. (J) The effect of NEDD4L on PIK3CA ubiquitination in HEK293T cells detected by IP assay. (K) The impact of NEDD4L on endogenous PIK3CA ubiquitination in HEK293T cells examined by IP assay. *p < 0.05 versus brain tissues from patients with non‐glioma or U937 cells transfected with oe‐NC. # p < 0.05 versus U937 cells transfected with sh‐NC. The experiment was repeated three times
FIGURE 6
FIGURE 6
NEDD4L downregulates PIK3CA to inhibit the PI3K/AKT pathway, thus restraining M2 polarization of macrophages and glioma cell proliferation, migration, and invasion. (A) The protein expression of NEDD4L, PIK3CA, and AKT and phosphorylated AKT in U937 cells after overexpression NEDD4L and PIK3CA examined by Immunoblotting. (B) The mRNA expression of iNOS, TNF‐α, Arg‐1, and IL‐10 in U937 cells after overexpression NEDD4L and PIK3CA measured by RT‐qPCR. (C) Immunoblotting of protein expression of iNOS and Arg‐1 in U937 cells after overexpression NEDD4L and PIK3CA. (D) The expression of TNF‐α and IL‐10 in supernatant of U937 cells after overexpression NEDD4L and PIK3CA assessed by ELISA. (E) The proportion of CD11b+CD163+ cells in U937 cells after overexpression NEDD4L and PIK3CA evaluated by flow cytometry. (F) The proliferation in U87 and A172 cells co‐cultivated with U937 cells tested by EdU. (G) Cell migration in U87 and A172 cells co‐cultivated with U937 cells examined by Transwell. (H) Cell invasion in U87 and A172 cells co‐cultivated with U937 cells examined by Transwell. *p < 0.05 versus U937 cells transfected with oe‐NC or U87 or A172 co‐cultivated with U937 cells treated without anything. # p < 0.05 versus U937 cells transfected with oe‐PIK3CA or U87 or A172 co‐cultivated with U937 cells transfected with oe‐PIK3CA. The experiment was repeated three times
FIGURE 7
FIGURE 7
Macrophage accelerates the formation of glioma in nude mice through hypoxic glioma‐derived EVs carrying miR‐10b‐5p. (A) Images of subcutaneous tumor formation after U937 treated with hypoxic glioma cell‐derived EVs, and U87 cells were transplanted into nude mice (n = 8). (B) Curves of subcutaneous tumor growth in nude mice transplanted with U937 cells treated with hypoxic glioma cell‐derived EVs and U87 cells (n = 8). (C) Tumor weight of tumor in nude mice transplanted with U937 cells treated with hypoxic glioma cell‐derived EVs and U87 cells presented by histogram (n = 8). (D) RT‐qPCR for determination of mRNA expression of miR‐10b‐5p, NEDD4L, PIK3CA (n = 8). (E) The protein expression of NEDD4L, PIK3CA, and AKT and phosphorylated AKT in tumor of nude mice transplanted with U937 cells treated with hypoxic glioma cell‐derived EVs and U87 cells determined by Immunoblotting (n = 8). (F) The expression of Ki67 in tumor of nude mice transplanted with U937 cells treated with hypoxic glioma cell‐derived EVs and U87 cells examined by Immunohistochemistry (n = 8) (×200). *p < 0.05 versus tumors of nude mice transplanted with U87 cells and U937 treated with PBS. # p < 0.05 versus tumors of nude mice transplanted with U87 cells and U937 treated with H‐EV‐mimic‐NC
FIGURE 8
FIGURE 8
Regulatory mechanism of hypoxic glioma‐derived EVs in glioma. Hypoxic glioma‐derived EVs harboring miR‐10b‐5p into macrophages reduced NEDD4L expression, thus diminishing the promotion of NEDD4L on the ubiquitylation and degradation of PIK3CA and increasing PIK3CA expression to activate the PI3K/AKT pathway. In this way, M2 polarization of macrophages was promoted, ultimately potentiating the proliferation, migration, and invasion of glioma cells
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