Research Paper Volume 13, Issue 7 pp 10415—10430

Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression

Exosomal miR-222 induce macrophages M2 polarization via targeting PTEN and activating Akt signaling pathway. (A) The predicted binding sites of miR-222 and PTEN on PicTar online database. (B) Luciferase reporter assay in cells transfected with wild-type or mutant PTEN 3’UTR as well as either miR-222 mimics or miR-222 inhibitors were performed. (C) Luciferase reporter assay in cells transfected with wild-type or mutant PTEN 3’UTR as well as either A/exo or S/exo were performed. (D) PTEN expression is negatively correlated with miR-222 level according to TCGA analysis. (E) Relative PTEN mRNA expressions in cells transfected with miR-222 mimics or miR-222 inhibitors were analyzed by PCR. (F) Relative PTEN protein expressions in cells transfected with miR-222 mimics or miR-222 inhibitors were analyzed by western blot. (G) Expressions of PTEN, tAkt, and pAkt in macrophages (M) treated with PBS, and S/exo or A/exo were analyzed by western blot. (H) Expressions of PTEN, tAkt, and pAkt in macrophages transfected with miR-222 mimics or miR-NC were analyzed by western blot. (I) Expressions of PTEN, tAkt, and pAkt in macrophages transfected with miR-222 inhibitors or miR-NC were analyzed by western blot. (J) Expressions of PTEN, tAkt, and pAkt in macrophages added with A/exo or transfected with miR-222 mimics were analyzed by western blot following Akt inhibitors treatment. Data are shown as mean ± SD, n = 3 independent experiments; * PPP

Figure 4. Exosomal miR-222 induce macrophages M2 polarization via targeting PTEN and activating Akt signaling pathway. (A) The predicted binding sites of miR-222 and PTEN on PicTar online database. (B) Luciferase reporter assay in cells transfected with wild-type or mutant PTEN 3’UTR as well as either miR-222 mimics or miR-222 inhibitors were performed. (C) Luciferase reporter assay in cells transfected with wild-type or mutant PTEN 3’UTR as well as either A/exo or S/exo were performed. (D) PTEN expression is negatively correlated with miR-222 level according to TCGA analysis. (E) Relative PTEN mRNA expressions in cells transfected with miR-222 mimics or miR-222 inhibitors were analyzed by PCR. (F) Relative PTEN protein expressions in cells transfected with miR-222 mimics or miR-222 inhibitors were analyzed by western blot. (G) Expressions of PTEN, tAkt, and pAkt in macrophages (M) treated with PBS, and S/exo or A/exo were analyzed by western blot. (H) Expressions of PTEN, tAkt, and pAkt in macrophages transfected with miR-222 mimics or miR-NC were analyzed by western blot. (I) Expressions of PTEN, tAkt, and pAkt in macrophages transfected with miR-222 inhibitors or miR-NC were analyzed by western blot. (J) Expressions of PTEN, tAkt, and pAkt in macrophages added with A/exo or transfected with miR-222 mimics were analyzed by western blot following Akt inhibitors treatment. Data are shown as mean ± SD, n = 3 independent experiments; * P<0.05, ** P<0.01, and *** P<0.001 compared with controls.