*3.3. MiR-21 Is Associated with GBM Tumorigenic Properties*

Next, we examined the effects of miR-21 on GBM cells by gene silencing and overexpression techniques. First, we demonstrated that miR-21-5p level increased in both U87MG and LN18 cells after being cocultured with GAM-derived exosomes (Figure 3A). We then transfected GBM (postincubation with GAM exosomes) with either mimic or inhibitor molecules of miR-21-5p. We found that the stemness markers Sox2, Oct4, Wnt, STAT3, and Nestin were all significantly increased when mimic miR-21-5p was added to both cells, while the opposite occurred after the miR-21-5p level was inhibited (Figure 3B). The Western blotting results agreed with the real-time PCR results (Figure 3C), where an increased miR-21-5p level led to the increased expression of Sox2, Oct4, STAT3, Akt, Nestin, and Wnt and a decreased level of GFAP. More importantly, tumorigenic properties such as colony formation and tumor sphere formation were also positively correlated with the level of miR-21-5p. For instance, an increased miR-21-5p level by mimic molecules led to an increased number of colonies (Figure 3D) and neurospheres (Figure 3E) generated, and the opposite was true with a decreased level of miR-21-5p with the inhibitor treatment. Furthermore, miR-21-5p mimic transfection made both U87MG and LN18 cells more resistant against TMZ, whereas miR-21-5p inhibitor reversed the resistance (Figure 3F).

**Figure 3.** GAM-derived exosomes promoted GBM tumorigenesis via miR-21-5p. (**A**) U87MG and LN18 cells incubated with GAM-derived exosomes showed a significantly increased level of miR-21-5p. GBM tumorigenesis was associated with miR-21-5p. Increased miR-21-5p level (by mimic molecules) in GBM cells showed an increased mRNA level of Sox2, Oct4, Wnt, STAT3, and Nestin or protein level of Sox2, Oct4, STAT3, Akt, Wnt, and Nestin with decreased GFAP, while a decrease in miR-21-5p (inhibitor) led to the opposite phenomenon (**B**,**C**). Incubation with GAM-derived exosomes increased colony-forming ability (**D**) and tumor-sphere-generating ability (**E**) in both U87MG and LN18 cells. (**F**) U87MG and LN18 cells transfected miR-21-5p mimic molecules (increased miR-21-5p level) resulted in significantly increased TMZ resistance, while there was reduced miR-21-59 and decreased TMZ resistance. Scale lengths = 100 μm, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
