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Research Paper Volume 11, Issue 15 pp 5300-5318
Exosomes derived from microRNA-199a-overexpressing mesenchymal stem cells inhibit glioma progression by down-regulating AGAP2
Relevance score: 5.649161Lei Yu, Si Gui, Yawei Liu, Xiaoyu Qiu, Guozhong Zhang, Xi’an Zhang, Jun Pan, Jun Fan, Songtao Qi, Binghui Qiu
Keywords: glioma, microRNA-199a, AGAP2, mesenchymal stem cells, exosomes, chemosensitivity, aging, gerontology, age-related-diseases
Published in Aging on August 5, 2019
miR-199a may regulate the AGAP2 gene in gliomas. (A) GO enrichment analysis on the differential genes in glioma-related profiles. The abscissa represents GO items and the ordinate represents the number of the differential genes. (B) KEGG functional enrichment analysis of the DEGs in glioma expression profile. The abscissa represents GeneRatio and the ordinate represents the KEGG items. The circle color and circle size indicate the p value and Count value, respectively. (C) the expression of AGAP2 gene in GSE79097 profile. The abscissa indicates the sample type and the ordinate indicates the gene expression. (D) the prediction of regulatory miRNAs of AGAP2. The four ellipses in the figure represent the prediction results from TargetScan database, mirDIP database and starBase database, and the expression of miRNAs in exosomes reported in the relevant literature. The middle part represents the intersection of four sets of data.
MiR-199a is poorly expressed, while AGAP2 highly expressed in glioma tissues and cell lines. (A) the expression of miR-199a in normal brain tissues and glioma tissues, determined by RT-qPCR. (B) the expression of miR-199a in NHAs and the glioma cell lines, determined by RT-qPCR. (C–D) the expression of AGAP2 in normal brain tissues and glioma tissues, detected by immunohistochemistry (200 ×). (E–F) the protein expression of AGAP2 in NHAs and different glioma cell lines, detected by Western blot analysis. * p < 0.05 compared with normal brain tissues or NHAs. The results were measurement data, presented as mean ± standard deviation. Independent sample t-test was used for the analysis in A and (D) One-way analysis of variance was used for other analyses. In tissues experiment, there were 75 glioma tissues and 17 normal brain tissues. The cell experiment was repeated three times.
Over-expression of miR-199a inhibits the migration, invasion and proliferation of U251 cells. (A–B) the migration ability of U251 cells measured by scratch test (Scale bar = 100 μM). (C–D) the invasion ability of U251 cells evaluated by Transwell assay (Scale bar = 50 μM). (E–F) the proliferation ability of U251 cells assessed by EdU assay (Scale bar = 50 μM). * p < 0.05 compared with the corresponding NC. The results were measurement data, presented as mean ± standard deviation. Comparison between two groups using independent sample t test. The experiment was repeated three times. mimic-NC, cells transfected with mimic-negative control; miR-199a mimic, cells transfected with miR-199a mimic; inhibitor-NC, cells transfected with inhibitor-negative control; miR-199a inhibitor, cells transfected with miR-199a inhibitor.
MiR-199a directly targets AGAP2 and down-regulates its expression. (A) the target binding site between miR-199a and AGAP2 was predicted through the bioinformatics website. (B) the results of the dual luciferase reporter gene assay. (C) the mRNA expression of AGAP2 in U251 and T98G cell lines with over-expressed or inhibited miR-199a, determined by RT-qPCR. (D–E) the protein expression of AGAP2 in U251 and T98G cell lines with over-expressed miR-199a, detected by Western blot analysis. (F) the protein expression of AGAP2 in U251 and T98G cell lines in the presence of the inhibition of miR-199a, detected by Western blot analysis. * p < 0.05 compared with the corresponding NC. The results were measurement data, presented as mean ± standard deviation. Paired t-test was used for comparison between two groups. The experiment was repeated three times. mimic-NC, cells transfected with mimic-negative control; miR-199a mimic, cells transfected with miR-199a mimic; inhibitor-NC, cells transfected with inhibitor-negative control; miR-199a inhibitor, cells transfected with miR-199a inhibitor.
The inhibitory role of miR-199a in U251 cells is mediated by the down-regulation of AGAP2. (A) the protein expression of AGAP2 after AGAP2 silencing, detected by Western blot analysis. (B) the migration ability of U251 cells after AGAP2 silencing, measured by scratch test (Scale bar = 100 μM). (C) the invasion ability of U251 cells after AGAP2 silencing, assessed by Transwell assay (Scale bar = 50 μM). (D) the proliferation ability of U251 cells after AGAP2 silencing, evaluated by EdU assay (Scale bar = 50 μM); (E) the protein expression of AGAP2 in the presence of miR-199a mimic or sh-AGAP2 or sh-NC, detected by Western blot analysis. (F) the migration ability of U251 cells (Scale bar = 100 μM), measured by scratch test. (G) the invasion ability of U251 cells (Scale bar = 50 μM), assessed by scratch test. (H) the proliferation ability of U251 cells, evaluated by EdU assay. (A–D) * p < 0.05 compared with the sh-NC group; (E–H) * p < 0.05 compared with mimic-NC + sh-NC group. # p < 0.05 compared with the miR-119a mimic group. The results were measurement data, presented as mean ± standard deviation. Paired t-test was used for the analysis in A, C and D. One-way analysis of variance was used for other analyses. The experiment was repeated three times. sh-NC, cells transfected with sh-negative control; sh-AGAP2, cells transfected with sh-AGAP2; mimic-NC + sh-NC, cells transfected with mimic-negative control and sh-negative control; miR-199a mimic, cells transfected with miR-199a mimic; miR-199a mimic + sh-AGAP2, cells transfected with miR-199a mimic and sh-AGAP2.
Exosomes are successfully isolated from the supernatant of hMSCs. (A) the characterization of surface antigens of hMSCs, detected by flow cytometry. (B–C) the adipogenic differentiation of hMSCs stained with Oil Red O. (C) the osteogenic differentiation of hMSCs stained with Alizarin red S. Green, arrows indicate positive cells (Scale bar = 50 μM). (D) the characterization of exosome structure under the electron microscope (Scar bar = 200 nm). (E) the identification of the exosome surface marker CD63 by Western blot assay. * p < 0.05. The results were measurement data, presented as mean ± standard deviation. Paired t-test was used for the analysis in E. The experiment was repeated three times.
The hMSCs deliver miR-199a to glioma cells by secreting exosomes. (A) the expression of miR-199a in the presence of MSC-miR-199a, MSC-miR-control and MSC-empty, determined by RT-qPCR. (B) miR-199a could be delivered to U251 glioma cells form hMSCs, verified by fluorescence microscopy detection (scar bar = 25 μM). (C) the expression of miR-199a in U251 cells after co-culture, determined by RT-qPCR. (D) the expression of AGAP in U251 cells after co-culture, determined by RT-qPCR. * p < 0.05 compared with the MSCs + mimic control group or the mimic-NC + DMSO group; # p < 0.05 compared with the miR-199a-Cy3 + DMSO group. The results were measurement data, presented as mean ± standard deviation. One-way analysis of variance was used for comparison among multiple groups. The experiment was repeated three times. RT-qPCR, reverse transcription quantitative polymerase chain reaction; MSCs, mesenchymal stem cells. mimic-NC + DMSO, cells treated with mimic-negative control and dimethyl sulphoxide; miR-199a-Cy3 + DMSO, cells treated with miR-199a-Cy3 and dimethyl sulphoxide; miR-199a-Cy3 + GW4869, cells treated with miR-199a-Cy3 and GW4869; miR-199a-Cy3 + DMA, cells treated with miR-199a-Cy3 and DMA.
MSCs-derived exosomal miR-199a inhibits the proliferation, invasion and migration of glioma cells. (A–B) the invasion ability of U251 cells, assessed by Transwell assay (Scale bar = 50 μM). (C–D) the migration ability of U251 cells, measured by scratch test (Scale bar = 100 μM). (E–F) the proliferation ability of U251 cells, evaluated by EdU assay (Scale bar = 50 μM). * p < 0.05. The results were measurement data, presented as mean ± standard deviation. Paired t-test was used for the analysis. The experiment was repeated three times. MSCs-mimic-NC, glioma cells co-cultured with mesenchymal stem cells and transfected with mimic-negative control; MSCs-miR-199a mimic, glioma cells co-cultured with mesenchymal stem cells and transfected with miR-199a mimic.
Combination of miR-199a and hMSCs delivering miR-199a enhances the chemosensitivity of glioma cells to TMZ. (A–B) the migration ability of U251 cells, assessed by scratch test (Scale bar = 100 μM). (C–D) the invasive ability of U251 cells, measured by Transwell assay (Scale bar = 50 μM). (E–F) the proliferation ability of U251 cells, evaluated by EdU assay. (G–H) the glioma cell apoptosis, detected by TUNEL staining (Scale bar = 50 μM). * p < 0.05 compared with the mimic-NC + TMZ group; # p < 0.05 compared with the TMZ group. The results were measurement data, presented as mean ± standard deviation. Paired t-test was used for the analysis. The experiment was repeated three times. TMZ, cells treated with Temozolomide; mimic-NC + TMZ, cells treated with mimic-negative control and Temozolomide; miR-199a mimic + TMZ, cells treated with miR-199a mimic and Temozolomide.
Combination of miR-199a treatment and hMSCs containing miR-199a inhibits tumor growth in vivo. (A) the tumor sizes in tumor-bearing nude mice after treatment with PBS, MSCs-mimic NC, MSCs-miR-199a mimic. (B) the tumor volume in tumor-bearing nude mice after treatment with PBS, MSCs-mimic NC, MSCs-miR-199a mimic. (C) the tumor weight in tumor-bearing nude mice after treatment with PBS, MSCs-mimic NC, MSCs-miR-199a mimic. (D–E) the changes of AGAP2 expression, detected by immunohistochemistry (Scale bar = 50 μM). (F) the apoptosis of glioma cells in tumor-bearing nude mice, detected by TUNEL staining (Scale bar = 25 μM). * p < 0.05 compared with the PBS group. The results were measurement data, presented as mean ± standard deviation. Repeated measures of variance of analysis was used for the analysis in B and one-way variance of analysis was used for the other analyses. The experiment was repeated three times. PBS, cells treated with phosphate buffered saline; MSCs-mimic NC, mesenchymal stem cells transfected with mimic negative control; MSCs-miR-199a mimic, mesenchymal stem cells transfected with miR-199a mimic.
MSCs-derived exosomes deliver miR-199a to glioma cells inhibiting the proliferation, migration and invasion of glioma cells, as well as enhancing the chemosensitivity by down-regulating AGAP2.