## 3 Results
### Hsp60 and Related miRNAs Levels in EVs  
The levels of Hsp60 and related miRNAs were assessed in the EVs isolated from the plasma of patients before and at various times after surgery. Hsp60 levels in EVs from GBM patients were successfully measured in all the samples, with the results showing high levels throughout (Figure 3A). The Hsp60 levels in EVs isolated from plasma of patient with atypical meningiomas (WHO G II) were also successfully measured and the results showed that the methods applied can distinguish variations in the chaperonin levels in different patients/situations. For instance, 7 days after surgery, the Hsp60 levels were significantly lower than before surgery, and at 30 days after surgery, a significant increase could be detected (Figure 3A1).  
TargetScan prediction revealed that the 3' UTR of HSP01 (the Hsp60 gene) contains a putative miR-1 and miR-206 binding site. Therefore, our attention was focused on  
Figure 2: EV characterization. Representative TEM (**A**) and AFM (**B**) images showing the typical characteristics of EVs isolated from the plasma of patients with GBM IV (left) and Meningioma II grade (right). (**C**) Table showing the size, measured by DLS, of the EVs isolated from blood of patients at different times of follow-up: BS, Before Surgery; AS1, 7 days After Surgery; AS2, 30 days After Surgery. (**D**) Evaluation by WB of EVs markers in the EVs from patients with GBM IV (left) and Meningioma II grade (right), at different times of follow-up.  
miR-1 and miR-206 (Figure 3B), which are predicted to regulate Hsp60 expression [45] (confirmed by experimental data [20; 21; 22; 23; 24]), and miR-663, considered an in vivo GBM prognostic biomarker [47]. MiR-16 level was used for the normalization of all miRNAs levels (Table 2). The methods applied measured miRNAs successfully and the results indicated that they can detect their quantitative variations. For example, in GBM EVs, the levels of miR-206 and miR-663 did not change during the period tested, whereas the level of miR-1 was increased 30 days after surgery in comparison with the levels at 7 days after surgery (Figure 3C). Furthermore, in meningiomas, miR1, miR-206, and miR-663 showed different levels at the various time points tested. miR-1 levels were low at 7 days after surgery but were again high at 30 days. In contrast, MiR-206 and miR-663 were low at 30 days after surgery (Figure 3C1).  
Figure 3: Measurement of Hsp60 and related miRNAs levels in EVs. Western blots and corresponding histograms showing the presence and levels of Hsp60 in EVs from patients with GBM IV (**A**) and in patients with meningioma II grade, at different times of follow-up (**A1**). BS, Before Surgery; AS1, 7 days After Surgery; AS2, 30 days After Surgery. Visible are the high levels of Hsp60 revealed by the standardized procedure in EVs from patients with GBM IV and from patients with meningioma II grade. In the latter, the method revealed differences of Hsp60 levels before and after surgery that were statistically significant (data are presented as the mean \(\pm\) S.D. * \(p<0.05\); \(\#\)\(p<0.01\)). (**B**) Predicted miR-1 and miR-206 binding sites detected in the HSPDI 3′ UTR region by TargetScan and the underlined base pairs indicate the target region we adopted. (**C**) Examples of results of our measurements with real-time PCR of the levels of miR1, miR-206, and miR-663 in EVs isolated from blood of GBM IV and from meningioma II grade patients (**C1**) The data in the horizontal histograms were normalized with the reference genes, according to the Livak method (\(2-\Delta\Delta\)CT). Data are presented as the mean \(\pm\) S.D. * \(p<0.05\); \(\#\)\(p<0.01\).