Fig 2: EZH2 contributes to miR-454-3p downregulation via DNA methylation to promote polarization of M2 macrophages. (A) The expression of miR-454-3p in clinical samples of different grades of glioma detected by RT-PCR. *p < 0.05 vs. normal brain tissues, #p < 0.05 vs. WHO II glioma tissues, &p < 0.05 vs. WHO III glioma tissues. (B) The efficiency of miR-454-3p and miRZIP-454 verified by RT-qPCR. *p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (C) Pearson correlation analysis of EZH2 and miR-454-3p expression in glioma clinical samples. (D) RT-qPCR to detect the expression of miR-454-3p and PTEN in glioma cells after EZH2 inhibition. *p < 0.05 vs. the treatment with Lenti-HK. (E) The proportion of CD11b+ CD206+ cells in cells after co-culture of miR-454-3p-overexpressed/knockout A172 cells with THP-1 cells detected by flow cytometry. (F) The expression of IL-8, IL-6, and MIP-3a in the macrophages after co-culture with A172 cells treated with Lenti-PTEN determined by RT-qPCR. (G) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3a in the tumor microenvironment. (H) The levels of IL-8, IL-6, and MIP-3a in the supernatant detected by ELISA. p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (I) CpG island and primer prediction. (J) MethPrime analysis of promoter CpG islands of miR-454-3p. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Pearson correlation analysis was performed to observe the correlation of indicators. Experiments were repeated three times independently.

Fig 3: PTEN expression is enhanced by miR-454-3p overexpression in glioma cells. (A) The efficiency of Lenti-PTEN determined using western blot analysis. (B) The protein expression of PTEN after PTEN knockdown. *p < 0.05 vs. treatment with Lenti-HK. (C) The proportion of CD11b+ CD206+ cells in the co-cultured cells detected by flow cytometry. (D) The expression of IL-8, IL-6, and MIP-3a in the macrophages after co-culture with A172 cells treated with Lenti-PTEN determined with RT-qPCR. (E) The expression of IL-8, IL-6, and MIP-3a in the tumor microenvironment detected using immunofluorescence. (F) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3a in the tumor microenvironment. (G) The levels of IL-8, IL-6, and MIP-3a in supernatant detected by ELISA. (H) Western blots of PTEN after treatment of miR-454-3p into glioma cell. (I) Western blot analysis of the protein expression changes of PTEN after treatment of miR-454-3p into glioma cells. (J) Western blots of PTEN after treatment of miRZIP-454. (K) Western blot analysis of the protein expression changes of PTEN after treatment of miRZIP-454 into glioma cells. *p < 0.05 vs. treatment with control-miR, #p < 0.05 vs. treatment with control-miRZIP. (L) The binding of miR-454-3p to PTEN after treatment of miRZIP-454 into glioma cells evaluated using a dual luciferase reporter system. (M) The binding of miR-454-3p to PTEN after treatment of miRZIP-454 into glioma cells evaluated using a dual luciferase reporter system. (N) The expression of PTEN mRNA every 4 h after treatment of miR-454-3p and addition of RNA synthesis inhibitor a-amanitin (50 mM) into glioma cells. (O) The expression of PTEN mRNA every 4 h after treatment of miRZIP-454 and addition of RNA synthesis inhibitor a-amanitin (50 mM) into glioma cells. *p < 0.05 vs. treatment with control-miR, #p < 0.05 vs. treatment with control-miRZIP. (P) YTHDF2 gene expression in glioma clinical specimens measured by RT-qPCR. *p < 0.05 vs. normal brain tissues, #p < 0.05 vs. WHO II glioma tissues, &p < 0.05 vs. WHO III glioma tissues. (Q) Detection of PTEN gene expression in glioma clinical specimens using RT-qPCR. *p < 0.05 vs. normal brain tissues, #p < 0.05 vs. WHO II glioma tissues, &p < 0.05 vs. WHO III glioma tissues. (R) Pearson correlation analysis of YTHDF2 and PTEN expression in glioma clinical samples. (S) Pearson correlation analysis of YTHDF2 and miR-454-3p expression in glioma clinical samples. (T) The YTHDF2 protein expression after overexpression of miR-454-3p measured by Western blot analysis. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Comparison of data at different time points was analyzed using two-way ANOVA. Pearson correlation analysis was performed to observe the correlation of indicators. Experiments were repeated three times independently.