*3.4. STAT3 and PDCD4 are Targets of miR-21-5p*

Subsequently, we examined the potential target(s) for miR-21-5p using bioinformatics tools and we identified STAT3, a well-known oncogene, and PDCD4, an established tumor suppressor, as the top-ranking candidates from all three algorithms used (PITA, miRmap, and miRanda). A potential site of interaction between miR-21-5p and STAT3 and PDCD4 was identified in the 3'UTR (upper panel, Figure 4A); more importantly, based on TCGA database, a strong negative correlation between the expression level of PDCD4 and miR-21-5p was established within a cohort of GBM patients (*n* = 525, lower panel, Figure 4B). We then demonstrated that increased miR-21-5p by mimic molecules in both U87MG and LN18 cells supported the negative correlation between the expression of miR-21-5p and PDCD4. Conversely, a decreased level of miR-21-5p by inhibitor molecules restored the expression of PDCD4 (Figure 4C). We then cocultured miR-21-5p-silenced GBM cells with GAMs and observed a significantly reduced M2 signature (CD68+/CD206+) (Figure 4D). More importantly, the M2 cytokines VEGF, TGF-β1, and IL-6 released by GAMs cocultured with miR-21-5p-silenced U87MG cells were significantly reduced and restored partially after cocultured miR-21-5p-silenced U87MG were transfected with mimic of miR-21-5p (Figure 4E).

**Figure 4.** MiR-21-5p targets STAT3 and PDCD4. (**A**) The bioinformatics tool shows miR-21-5p binding to the 3'UTR of STAT3 and PDCD4 (upper panel). (**B**) A negative correlation between the expression of miR-21-5p and PDCD4 in the GBM database (*n* = 525, TCGA). (**C**) An increased miR-21-5p level (by mimic molecule, lane M) led to significantly reduced PDCD4 expression in both U87MG and LN18 cells; the reverse was true with the inhibitor of miR-21-5p. (**D**) Flow cytometry analysis showed a significantly reduced CD206+/CD68<sup>+</sup> population in GAMs cocultured with miR-21-5p-silenced U87MG and LN18 cells; the reverse was observed in miR-21-5p mimic transfected coculture experiments. (**E**) The inhibitor of miR-21-5p resulted in the reduction of VEGF, TGF- β1, and IL-6 secreted by the U87MG cells into the culture medium. \*\*\* *p* < 0.001.
